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Biomarkers in colorectal cancer (CRC) are of great interest in the current literature due to improvements in techniques such as liquid biopsy and next-generation sequencing (NGS). However, screening methods vary globally, with multi-target stool DNA (mt-sDNA) predominantly used in the USA and, more recently, the Cologuard Plus; biomarkers such as the Galectins family and septins show promise in early detection. Gut microbiome assessments, such as Fusobacterium nucleatum, are under intense exploration. Diagnostic tests, such as circulating DNA analysis via NGS, exhibit effectiveness and are being increasingly adopted. Circulating tumor cells emerge as potential alternatives to traditional methods in terms of diagnosis and prognosis. Predictive biomarkers are well established in guidelines; nonetheless, with the aid of machine learning and artificial intelligence, these biomarkers may be improved. This review critically explores the actual dynamic landscape of CRC biomarkers and future, promising biomarkers involved in screening, diagnosis, and prognosis. [ABSTRACT FROM AUTHOR]

Simple Summary: This study compared a no-touch technique with conventional radical nephrectomy for the treatment of renal cell carcinoma in terms of circulating tumor cell (CTC) and circulating mesenchymal cell (CMC) release and patient prognosis. The results showed that the no-touch approach did not reduce CTC or CMC release or improve survival, suggesting that early pedicle ligation, the last standing Robson principle of radical nephrectomy, may have fallen. Nonetheless, the no-touch technique proved to be faster and as safe as the conventional radical nephrectomy. Healthy controls had no circulating cells; however, high CMC counts were found in chronic inflammation controls and oncocytoma patients, with no difference to RCC patients. Background: Circulating tumor cells (CTCs) may be the missing renal cell carcinoma (RCC) biomarker. No-touch (NT) resection has shown benefit in several tumors. Methods: A randomized controlled trial comparing CTC and circulating mesenchymal cell (CMC) release in no-touch (NT) vs. conventional (C) laparoscopic RN. Blood samples were collected at operation room arrival (S0), specimen extraction (S1), postoperative D1, and D30. CTCs were isolated and analyzed using RUBYchip™. Results: Thirty-four patients were included. No significant differences were found between groups in CTC and CMC counts, count variations between time points, complications, and survival. The total circulating cell detection rates in the NT, C, and overall RCC groups were 58.3%, 80.0%, and 70.4% at S0; 41.6%, 86.7%, and 66.7% at S1; 50.0%, 64.3%, and 60.0% at D1; and 54.5%, 42.9%, and 44.0% at D30, respectively. A progressive decrease in CMCs was observed in the C group after surgery, especially at D1 (4.78 to 1.64 CMCs/7.5 mL blood, p = 0.035). Healthy controls had no circulating cells; however, high CMC counts were found in chronic inflammation controls and oncocytoma patients, with no significant difference from RCC patients (p = 0.460). Conclusions: NT RN did not reduce circulating cell release nor improve survival compared to C RN. [ABSTRACT FROM AUTHOR]

Cancer cell clusters have a higher capacity for metastasis than single cells, suggesting cancer cell clusters have biological properties different from those of single cells. The nature of de novo cancer cell clusters that are newly formed from tumor masses is largely unknown. Herein, we generated small cell clusters from colorectal cancer organoids and tracked the growth patterns of the clusters up to four cells. Growth patterns were classified into actively growing and poorly growing spheroids (PG). Notch signaling was robustly activated in small clusters immediately after dissociation, and Notch signaling inhibition markedly increased the proportion of PG spheroids. Only a limited number of PG spheroids grew under growth‐permissive conditions in vitro, but xenograft tumors derived from Notch inhibited clusters showed growth rates comparable to those of untreated spheroids. Thus, de novo clusters are composed of cells with interchangeable growth fates, which are regulated in a context‐dependent manner by Notch signaling. [ABSTRACT FROM AUTHOR]

Background: We developed a Rare Cell Sorter (RCS) for collecting single cell including circulating tumor cells (CTCs). This single-institution pilot study evaluated the ability of this device to detect tumor-like cells in patients with lung cancer and confirmed their genuineness based on the epidermal growth factor receptor (EGFR) mutation concordance with tissue samples. Methods: This study included patients treated for lung cancer from September 2021 to August 2022 in University of Tsukuba Hospital. Peripheral blood samples were obtained before surgery or during periodic medical checks for patients treated with drugs. We used the RCS to capture cells based on size. The cells were stained, and the Hoechst-positive, CD45-negative, and epithelial celladhesion molecule (EpCAM)- positive cells were defined as CTCs, were collected. The presumptive CTCs were counted and tested using digital droplet polymerase chain reaction for EGFR mutations and compared with the tissue EGFR status to check concordance. Results: Eighteen patients were included in this study and CTCs were detected in 6 patients (33%). The CTCs from three patients showed EGFR mutation, and the EGFR mutation status of CTCs concorded with that of tissue samples in 83% of the cases (5/6). Only one CTC showed a different status from the tissue, and the concordance rate of EGFR status between CTCs and the tissue was 96% (24/25). Conclusion: The ability of the RCS to detect CTCs in patients with lung cancer was demonstrated based on the concordance of EGFR status in this pilot study. This novel hybrid method of CTC recovery using the RCS has the potential to recover a wide range of CTCs regardless of EpCAM. Further validation through a large-scale study is needed. [ABSTRACT FROM AUTHOR]

Background: The potential value of detecting epithelial–mesenchymal transition (EMT) CTCs in early breast cancer, especially during the neoadjuvant therapy period, requires further investigation. We analyzed dynamic CTC phenotype status, to improve recurrence risk stratification for patients with stage III breast cancers. Methods: We enrolled 45 patients with stage III breast cancers from 2 clinical trials undergoing neoadjuvant chemotherapy and utilized the CanPatrol CTC enrichment technique pre- and post-chemotherapy to identify CTC phenotypes, including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs, in peripheral blood samples. Kaplan–Meier analyses were conducted to explore the prognostic value of dynamic change of CTC count and the proportion of CTCs with different phenotypes. Then, redefine the risk stratification based on CTC status and clinicopathological risk in combination. Results: Increased proportion of M + CTCs was a high-risk CTC status that was associated with decreased DFS (HR, 3.584; 95% CI, 1.057–12.15). In a combined analysis with clinicopathological risk, patients with high-risk tumors had an elevated risk of recurrence compared to patients with low-risk tumors (HR, 4.482; 95% CI, 1.246–16.12). The recurrence risk could be effectively stratified by newly defined risk stratification criteria, with 5-year DFS of 100.0%, 77.3%, and 50.0%, respectively, for low-risk, mid-risk, and high-risk patients (P = 0.0077). Finally, in the ROC analysis, the redefined risk stratification demonstrated higher predictive significance with an AUC of 0.7727, compared to CTC status alone (AUC of 0.6751) or clinicopathological risk alone (AUC of 0.6858). Conclusion: The proportion of M + CTCs increased after neoadjuvant chemotherapy indicating a higher risk of tumor recurrence. Combining CTC status with clinicopathological risk has potential to redefine the risk stratification of stage III breast cancers and provide improved predictions of relapse. [ABSTRACT FROM AUTHOR]

Detecting circulating tumor cells has exhibited great significance in treating cancers since its concentration is an index strongly associated with the development and transfer of the tumor. However, the present commercial method for CTC detection is still expensive, because special antibodies and complicated devices must be used for cell separation and imaging. Hence, it is quite necessary to apply alternative materials and methods to decrease the cost of CTC detection. In this article, we coated a cellulose acetate membrane with nanoparticles formed by the polymerization of melamine and furfural, creating a surface with nanoscale roughness for the highly efficient capture of the sparse CTCs in a blood sample. Subsequently, the CTCs on the surface can be quantitatively detected by colorimetry with the aid of a COF-based nanozyme. The detection limit (LOD) can be as low as 3 cells/mL, which is the lowest LOD among the colorimetric methods to our knowledge. Considering the low cost of fabricating the membrane for CTC capture and the robustness of nanozymes compared with natural enzymes, this CTC detection approach displays great potential to decrease the financial burden of commercial CTC detection. [ABSTRACT FROM AUTHOR]

Objective: To construct a recombinant adenovirus (Ad-DF3-copGFP) containing the DF3/MUC1 promoter transcriptional regulatory sequence and green fluorescent protein (GFP) and to investigate its role in the detection of circulating tumor cells (CTC). Methods: A recombinant adenovirus (Ad - DF3 - copGFP) was prepared and purified. Comparative studies were conducted with a previously constructed and stored recombinant adenovirus (Ad-hTERT-copGFP) in our laboratory. Infection efficiency and non-specific infection rates were evaluated by infecting lung adenocarcinoma A549 and H1299 cells, as well as peripheral blood mononuclear cells (PBMC)from healthy individuals. A549 cells were artificially added to healthy peripheral blood to simulate CTC, and the infection with both recombinant viruses was used to detect simulate CTC for detection rate determination. The Ad-DF3-copGFP and Ad-hTERT- copGFP methods were used to detect CTC in clinical samples from lung cancer patients, and the initial clinical CTC detection performance was evaluated. Results: The recombinant adenovirus (Ad -DF3 - copGFP) was successfully constructed, showing a high infection efficiency for both A549 and H1299 cells. Compared with Ad-hTERT-copGFP, Ad-DF3-copGFP showed a lower non-specific infection rate (P < 0.001). The overall detection rate using the Ad-DF3-copGFP method (77.3%) was higher than that using the AdhTERT-copGFP method (69.6%). In clinical CTC detection, the number of CTC detected by the Ad-DF3-copGFP method [(10.90± 2.42) cells per 4 mL]was significantly higher than that by the Ad-hTERT-copGFP method [(6.20±1.81) cells per 4 mL, P < 0.001]. Conclusion: The recombinant adenovirus (Ad - DF3 - copGFP) is successfully constructed, demonstrating a reliable and efficient detection of CTC, thus providing a new method for CTC detection. [ABSTRACT FROM AUTHOR]

Neutrophil extracellular traps (NETs) are fibrous web-like structures composed of decondensed chromatin and granular proteins released by neutrophils, with the ability to capture and kill bacteria. Pathogens, such as bacteria and viruses, can trigger the formation of NETs via NETosis, a type of programmed cell death that has two distinct forms: suicidal NETosis and vital NETosis. Numerous studies have found that NETs interact with immune cells in the tumor microenvironment, where they activate macrophages, promote immunosuppressive effects of myeloid-derived suppressor cells, and coat the tumor surface to prevent cytotoxic effects of CD8+ T cells and natural killer cells. Recent research has identified a substantial presence of NETs in oral squamous cell carcinoma (OSCC) tissues, indicating a complex relationship between NETs and OSCC development. Depending on the phenotype of neutrophils, NETs may exhibit pro-tumor or anti-tumor effects. For instance, NETs derived from N1-type neutrophils may exert anti-tumor effects, while TGF-β-induced NETs derived from N2-type neutrophils may exert pro-carcinogenic activity, thereby contributing to the development of oral squamous metaplasia. Furthermore, NETs likely play a role in OSCC metastasis by capturing circulating tumor cells and inducing a hypercoagulable state, thereby facilitating tumor-related thrombus formation and hematogenous metastasis. The involvement of NETs in the occurrence and progression of OSCC opens new avenues for anti-tumor therapy and prognostication. Inhibiting NET formation can significantly suppress the development of chemotherapy-induced drug resistance and reduce the risk of thrombosis in OSCC patients, thereby inhibiting tumor metastasis. Currently, multiple prognostic models based on NET-related genes have been constructed and validated for head and neck squamous cell carcinoma, indicating the potential clinical value of NETs. However, the association between NETs and OSCC treatment is still unclear, necessitating further research on its underlying mechanisms and feasibility. This article attempts to review the relationship between NETs and OSCC, aiming to provide novel perspectives for OSCC treatment.

Ming Yu, Jiaqin Deng, Yihua Gu, Yeqian Lai Department of Thyroid Surgery, Meizhou People’s Hospital, Meizhou Academy of Medical Sciences, Meizhou, People’s Republic of ChinaCorrespondence: Ming Yu, Department of Thyroid Surgery, Meizhou People’s Hospital, Meizhou, People’s Republic of China, Email 13543234215@163.comObjective: Circulating tumor cell (CTC) has been used to assist in the diagnosis and progression assessment of solid tumors, but the relationship between preoperative CTCs levels and central lymph node metastasis (CLNM) of papillary thyroid carcinoma (PTC) needs to be clarified.Methods: Data on clinical features (age, gender, Hashimoto’s thyroiditis, multifocal, maximum lesion diameter, invaded capsule, clinical stage, and status of lymph node metastasis) of PTC patients treated at our hospital between June 2021 and April 2023 were retrospectively collected. The relationship between the CTCs level and these clinical features was analyzed, especially the relationship between the CTCs level and CLNM.Results: A total of 705 PTC patients were included, and there were 333 (47.2%) patients with CLNM. Patients with a high CTCs level had higher proportions of multifocality, maximum lesion diameter > 1cm, and CLNM than those in patients with a low CTCs level. Tumor size was connected to CTCs level, patients with a high CTCs level had a higher proportion of CLNM than those with a low CTCs level in PTC with maximum lesion diameter ≤ 1cm (45.3% vs 29.7%) (p=0.001). Logistic regression analysis showed that age < 55 years old (odds ratio (OR): 2.612, 95% confidence interval (CI): 1.565– 4.361, p< 0.001), invaded capsule (OR: 1.662, 95% CI: 1.098– 2.517, p=0.016), and high CTCs level (≥ 8.7 FU/3mL, OR: 2.141, 95% CI: 1.431– 3.203, p< 0.001) were associated with CLNM in PTC with maximum lesion diameter ≤ 1cm.Conclusion: In PTC patients with maximum lesion diameter ≤ 1cm, patients with high preoperative CTCs level (≥ 8.7FU/3mL), age < 55 years old, and capsular invasion were prone to CLNM. However, similar results were not observed in patients with maximum lesion diameter > 1cm.Keywords: papillary thyroid carcinoma, circulating tumor cell, central lymph node metastasis, lymph node metastasis

Abstract Background We developed a Rare Cell Sorter (RCS) for collecting single cell including circulating tumor cells (CTCs). This single-institution pilot study evaluated the ability of this device to detect tumor-like cells in patients with lung cancer and confirmed their genuineness based on the epidermal growth factor receptor (EGFR) mutation concordance with tissue samples. Methods This study included patients treated for lung cancer from September 2021 to August 2022 in University of Tsukuba Hospital. Peripheral blood samples were obtained before surgery or during periodic medical checks for patients treated with drugs. We used the RCS to capture cells based on size. The cells were stained, and the Hoechst-positive, CD45-negative, and epithelial celladhesion molecule (EpCAM)- positive cells were defined as CTCs, were collected. The presumptive CTCs were counted and tested using digital droplet polymerase chain reaction for EGFR mutations and compared with the tissue EGFR status to check concordance. Results Eighteen patients were included in this study and CTCs were detected in 6 patients (33%). The CTCs from three patients showed EGFR mutation, and the EGFR mutation status of CTCs concorded with that of tissue samples in 83% of the cases (5/6). Only one CTC showed a different status from the tissue, and the concordance rate of EGFR status between CTCs and the tissue was 96% (24/25). Conclusion The ability of the RCS to detect CTCs in patients with lung cancer was demonstrated based on the concordance of EGFR status in this pilot study. This novel hybrid method of CTC recovery using the RCS has the potential to recover a wide range of CTCs regardless of EpCAM. Further validation through a large-scale study is needed.

Abstract Background Traditional genomic profiling and mutation analysis of single cells like Circulating Tumor Cells (CTCs) fails to capture post-translational and functional alterations of proteins, often leading to limited treatment efficacy. To overcome this gap, we developed a miniaturized ‘protein analysis on the single cell level’ workflow—baptized ZeptoCTC. It integrates established technologies for single-cell isolation with sensitive Reverse Phase Protein Array (RPPA) analysis, thus enabling the comprehensive assessment of multiple protein expression and activation in individual CTCs. Methods The ZeptoCTC workflow involves several critical steps. Firstly, individual cells are labeled and isolated. This is followed by cell lysis and the printing of true single cell lysate preparations onto a ZeptoChip using a modified micromanipulator, CellCelector™. The printed lysates then undergo fluorescence immunoassay RPPA protein detection using a ZeptoReader. Finally, signal quantification is carried out with Image J software, ensuring precise measurement of multiple protein levels. Results The efficacy of ZeptoCTC was demonstrated through various applications. Initially, it was used for measuring EpCAM protein expression, a standard marker for CTC detection, revealing higher levels in single MCF-7 over MDA-MB-231 tumor cells. Furthermore, in Capivasertib (Akt-inhibitor)-treated MCF-7 single cells, ZeptoCTC detected a 2-fold increase in the pAkt/Akt ratio compared to control cells, and confirmed co-performed bulk-cell western blot analysis results. Notably, when applied to individual CTCs from metastasized breast cancer patients, ZeptoCTC revealed significant differences in protein activation levels, particularly in measured pAkt and pErk levels, compared to patient-matched WBCs. Moreover, it successfully differentiated between CTCs from patients with different Akt1 genotypes, highlighting its potential to determine the activation status of druggable cancer driving proteins for individual and targeted treatment decision making. Conclusions The ZeptoCTC workflow represents a valuable tool in single cell cancer research, crucial for personalized medicine. It permits detailed analysis of key proteins and their activation status of targeted, cancer-driven signaling pathways in single cell samples, aiding in understanding tumor response, progression, and treatment efficacy beyond bulk analysis. The method significantly advances clinical investigations in cancer, improving treatment precision and effectiveness. The workflow will be applicable to protein analysis on other types of single cells like relevant in stem cell, neuropathology and hemopoietic cell research.

The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) is the standard-of-care for estrogen receptor (ER)-positive, HER2-negative (ER+/HER2− advanced/metastatic breast cancer (mBC). However, the impact of CDK4/6i on circulating immune cells and circulating tumor cells (CTCs) in patients receiving CDK4/6i and ET (CDK4/6i+ET) remains poorly understood. This was a prospective cohort study including 44 patients with ER+/HER2− mBC treated with CDK4/6i+ET in either first or second line. Peripheral blood samples were collected before (baseline) and 3 months (t2) after therapy. Immune cell's subsets were quantified by flow cytometry, and microfluidic-captured CTCs were counted and classified according to the expression of cytokeratin and/or vimentin. Patients were categorized according to response as responders (progression-free survival [PFS] ≥ 6.0 months; 79.1%) and non-responders (PFS < 6.0 months; 20.9%). CDK4/6i+ET resulted in significant changes in the hematological parameters, including decreased hemoglobin levels and increased mean corpuscular volume, as well as reductions in neutrophil, eosinophil, and basophil counts. Specific immune cell subsets, such as early-stage myeloid-derived suppressor cells, central memory CD4+ T cells, and Vδ2+ T cells expressing NKG2D, decreased 3 months after CDK4/6i+ET. Additionally, correlations between the presence of CTCs and immune cell populations were observed, highlighting the interplay between immune dysfunction and tumor dissemination. This study provides insights into the immunomodulatory effects of CDK4/6i+ET, underscoring the importance of considering immune dynamics in the management of ER+/HER2− mBC. [ABSTRACT FROM AUTHOR]

An elevated serum β2-microglobulin (β2M) level is indicative of impaired glomerular filtration and prerenal diseases, such as malignant tumors, autoimmune disorders, and liver diseases. An elevated serum β2M level has been shown to promote metastasis via the induction of epithelial–mesenchymal transition (EMT) in cancer cells. However, the therapeutic potential of targeting β2M remains unclear. Here, we aimed to investigate the efficacy of Filtor, a small polymethyl methacrylate fiber-based β2M removal column, in reducing the β2M level and suppressing cancer cell-induced EMT and metastasis. We assessed the effects of Filtor on the changes in metastasis based on the number of circulating tumor cells (CTCs), which reflects the post-EMT cancer cell population. We performed therapeutic apheresis using Filtor on a male patient with sinonasal neuroendocrine carcinoma, a female patient with a history of colorectal cancer, and another female patient with a history of pancreatic ductal adenocarcinoma. Significantly low serum β2M levels and CTC counts were observed immediately and 4 weeks after treatment compared with those in the pretreatment phase. Moreover, the CTC count immediately after therapeutic intervention was markedly reduced, likely because Filtor had trapped CTCs directly. These findings suggest that therapeutic apheresis with Filtor can prevent cancer metastasis and recurrence by directly removing CTCs. [ABSTRACT FROM AUTHOR]

Background: This study investigated the prevalence and subtype distribution of circulating tumor cells (CTCs) in patients with papillary thyroid cancer (PTC) before and after thyroidectomy to determine the potential of CTC count as a noninvasive marker of the efficacy of surgical treatment in PTC. Materials and methods: Between January 2021 and January 2022, 62 PTC patients who underwent thyroidectomy at Seoul National University Bundang Hospital were prospectively evaluated. Peripheral blood samples (7.5 ml) were collected from each patient for CTC analysis before surgery and at 2 weeks and 3 months after surgery. CTC count and the distribution of CTC subtypes, including epithelial, epithelial-mesenchymal, and mesenchymal phenotypes, were analyzed using the negative selection method and immunofluorescence staining. The relationship between CTC count and clinicopathological characteristics was analyzed before and after surgery. Results: Before surgery, CTCs were detected in 87% (54/62) of patients; the mean CTC count was 8.0 and the median was 5.0 in 7.5 ml of peripheral blood. The mesenchymal or epithelial-mesenchymal phenotypes were predominant. After thyroidectomy, the mean and median CTC count values decreased to 5.3 and 2.5, respectively, at 2 weeks and to 4.3 and 3.0, respectively, at 3 months. This postoperative reduction in CTCs was more pronounced in patients with lymphatic invasion, lymph node metastasis, or BRAF V600E mutation. Conclusion: CTCs were detected in patients with PTC with a predominance of cells undergoing epithelial-mesenchymal transition. The CTC count decreased postoperatively, suggesting that liquid biopsy with CTC detection could be a valuable noninvasive tool for monitoring the efficacy of surgery in PTC. [ABSTRACT FROM AUTHOR]

Currently, tumor treatment modalities such as immunotherapy and targeted therapy have more stringent requirements for obtaining tumor growth information and require more accurate and easy‐to‐operate tumor information detection methods. Compared with traditional tissue biopsy, liquid biopsy is a novel, minimally invasive, real‐time detection tool for detecting information directly or indirectly released by tumors in human body fluids, which is more suitable for the requirements of new tumor treatment modalities. Liquid biopsy has not been widely used in clinical practice, and there are fewer reviews of related clinical applications. This review summarizes the clinical applications of liquid biopsy components (e.g., circulating tumor cells, circulating tumor DNA, extracellular vesicles, etc.) in tumorigenesis and progression. This includes the development process and detection techniques of liquid biopsies, early screening of tumors, tumor growth detection, and guiding therapeutic strategies (liquid biopsy‐based personalized medicine and prediction of treatment response). Finally, the current challenges and future directions for clinical applications of liquid biopsy are proposed. In sum, this review will inspire more researchers to use liquid biopsy technology to promote the realization of individualized therapy, improve the efficacy of tumor therapy, and provide better therapeutic options for tumor patients. [ABSTRACT FROM AUTHOR]

A highly sensitive, circulating tumor cell (CTC)-based liquid biopsy was used to monitor gastrointestinal cancer patients during treatment to determine if CTC abundance was predictive of disease recurrence. The approach used a combination of biomimetic cell rolling on recombinant E-selectin and dendrimer-mediated multivalent immunocapture at the nanoscale to purify CTCs from peripheral blood mononuclear cells. Due to the exceptionally high numbers of CTCs captured, a machine learning algorithm approach was developed to efficiently and reliably quantify abundance of immunocytochemically-labeled cells. A convolutional neural network and logistic regression model achieved 82.9% true-positive identification of CTCs with a false positive rate below 0.1% on a validation set. The approach was then used to quantify CTC abundance in peripheral blood samples from 27 subjects before, during, and following treatments. Samples drawn from the patients either prior to receiving radiotherapy or early in chemotherapy had a median 50 CTC ml-1 whole blood (range 0.6-541.6). We found that the CTC counts drawn 3 months post treatment were predictive of disease progression (p = .045). This approach to quantifying CTC abundance may be a clinically impactful in the timely determination of gastrointestinal cancer progression or response to treatment.

Abstract Background Octamer-binding transcription factor 4-positive circulating tumor cell (OCT4+CTC) exhibits high stemness and invasive potential, which may influence the efficacy of immune checkpoint inhibitors (ICI). This study aimed to assess the prognostic role of OCT4+CTC in advanced cholangiocarcinoma (CCA) patients who received ICI treatment. Methods In total, 40 advanced CCA patients who received ICI treatment were included, and CTC and OCT4 counts were detected via a Canpatrol system and an RNA in situ hybridization method before ICI treatment. Patients were subsequently divided into none CTC, OCT4−CTC, and OCT4+CTC groups. Patients were followed up for a median of 10.4 months. Results The percentages of patients in none CTC, OCT4−CTC, and OCT4+CTC groups were 25.0%, 30.0%, and 45.0%, respectively. The proportion of patients with lymph node metastasis was highest in OCT4+CTC group, followed by none CTC group, and lowest in OCT4−CTC group (P = 0.025). The objective response rate (ORR) was lowest in OCT4+CTC group, moderate in OCT4−CTC group, and highest in none CTC group (P = 0.009), while disease control rate was not different among three groups (P = 0.293). In addition, progression-free survival (PFS) (P

Simple Summary: Hepatocellular carcinoma (HCC) represents a significant health challenge, demanding innovative approaches for effective treatment. Despite advances in immune checkpoint inhibitor (ICI) therapy, reliable biomarkers to predict treatment response in HCC have yet to be found. Our study addressed this critical research gap by exploring the role of circulating tumor cells (CTCs) and their PD-L1 RNA expression in predicting response to atezolizumab and bevacizumab (Atezo/Bev) treatment. CTC-derived RNA collected serially during Atezo/Bev treatment suggests that patients with higher levels of PD-L1 expression in CTCs at baseline were more responsive to this treatment, but not lenvatinib treatment. Moreover, PD-L1 RNA levels in CTCs were an accurate response predictor and a monitorable biomarker that changed dynamically to reflect the response during Atezo/Bev treatment. The results of this study highlight the potential for individualized treatment decisions based on PD-L1 expression in CTCs and have implications for clinical practice. There remains no reliable biomarker of therapeutic efficacy in hepatocellular carcinoma (HCC) for the PD-L1 inhibitor atezolizumab and bevacizumab (Atezo/Bev). Circulating tumor cells (CTCs) enable the serial collection of living tumor cells. Pre-treatment and serial CTC gene expression changes and tumor histology were evaluated to identify predictors of response to Atezo/Bev. Peripheral blood from 22 patients with HCC treated with Atezo/Bev and 24 patients treated with lenvatinib was serially collected. The RNA expression in CTCs was analyzed using qRT-PCR. Higher PD-L1 expression in pre-treatment CTCs was associated with response and improved prognosis with Atezo/Bev treatment, but not with lenvatinib. There was no correlation between PD-L1 expression in CTCs and that in liver tumor biopsy specimens scored using imaging software. Furthermore, PD-L1 RNA expression in CTCs was dynamically altered by Atezo/Bev, decreasing during effective response and increasing upon progression. CTC-derived RNA collected during Atezo/Bev indicates that patients with higher PD-L1 expression in CTCs at baseline were 3.9 times more responsive to treatment. Therefore, PD-L1 RNA levels in CTCs are an accurate response predictor and may be a monitorable biomarker that changes dynamically to reflect the response during Atezo/Bev treatment. [ABSTRACT FROM AUTHOR]

Background: Patients with breast cancer (BC) at advanced stages have poor outcomes because of high rate of recurrence and metastasis. Biomarkers for predicting prognosis remain to be explored. This study aimed to evaluate the relationships between circulating tumor cells (CTCs) and outcomes of BC patients. Patients and methods: A total of 50 female were enrolled in this study. Their diagnoses were determined by clinical characteristics, image data, and clinical pathology. CTC subtypes and TOP2A gene expression on CTCs were detected by CanPatrol™ technology and triple color in situ RNA hybridization (RNA-ISH), which divided into epithelial CTCs (eCTCs), mesenchymal CTCs (MCTCs), and hybrid CTCs (HCTCs) based on their surface markers. Hormone receptor, including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) expression, was measured by immunohistochemistry (IHC) method before treatment. The risk factors for predicting recurrence and metastasis were calculated by COX risk regression model. The progression-free survival (PFS) of patients was determined using Kaplan–Meier survival curve. Results: The patients with a large tumor size (≥ 3 cm) and advanced tumor node metastasis (TNM) stages had high total CTCs (TCTCs) (P < 0.05). These patients also had high TOP2A expression level. COX risk regression analysis indicated that TOP2A expression levels in TCTCs, ER + , HER-2 + , and TNM stages were critical risk factors for recurrence and metastasis of patients (P < 0.05). The PFS of patients with ≥ 5 TCTCs, ≥ 3 HCTCs, and positive TOP2A expression in ≥ 3 TCTCs was significantly longer than that in patient with < 5 TCTCs, < 3 HCTCs, and TOP2A expression in < 3 TCTCs (P < 0.05). In contrast, the PFS of patients with positive hormone receptors (ER + , PR + , HER-2 +) also was dramatically lived longer than that in patients with negative hormone receptor expression. Conclusions: High TCTC, HCTCs, and positive TOP2A gene expression on CTCs were critical biomarkers for predicting outcomes of BC patients. Positive hormone receptor expression in BC patients has significant favor PFS. [ABSTRACT FROM AUTHOR]

Background: Octamer-binding transcription factor 4-positive circulating tumor cell (OCT4+CTC) exhibits high stemness and invasive potential, which may influence the efficacy of immune checkpoint inhibitors (ICI). This study aimed to assess the prognostic role of OCT4+CTC in advanced cholangiocarcinoma (CCA) patients who received ICI treatment. Methods: In total, 40 advanced CCA patients who received ICI treatment were included, and CTC and OCT4 counts were detected via a Canpatrol system and an RNA in situ hybridization method before ICI treatment. Patients were subsequently divided into none CTC, OCT4−CTC, and OCT4+CTC groups. Patients were followed up for a median of 10.4 months. Results: The percentages of patients in none CTC, OCT4−CTC, and OCT4+CTC groups were 25.0%, 30.0%, and 45.0%, respectively. The proportion of patients with lymph node metastasis was highest in OCT4+CTC group, followed by none CTC group, and lowest in OCT4−CTC group (P = 0.025). The objective response rate (ORR) was lowest in OCT4+CTC group, moderate in OCT4−CTC group, and highest in none CTC group (P = 0.009), while disease control rate was not different among three groups (P = 0.293). In addition, progression-free survival (PFS) (P < 0.001) and overall survival (OS) (P = 0.001) were shorter in the OCT4+CTC group than in none CTC & OCT4−CTC group. Moreover, OCT4+CTC (versus none CTC) was independently linked with poorer PFS [hazard ratio (HR) = 6.752, P = 0.001] and OS (HR = 6.674, P = 0.003) in advanced CCA patients. Conclusion: OCT4+CTC relates to lymph node metastasis and shows a good predictive value for poor treatment response and survival in advanced CCA patients who receive ICI treatment. [ABSTRACT FROM AUTHOR]

Small cell lung cancer (SCLC), a highly aggressive malignancy, is rapidly at an extensive stage once diagnosed and is one of the leading causes of death from malignancy. In the past decade, the treatment of SCLC has largely remained unchanged, and chemotherapy remains the cornerstone of SCLC treatment. The therapeutic value of adding immune checkpoint inhibitors to chemotherapy for SCLC is low, and only a few SCLC patients have shown a response to immune checkpoint inhibitors. Circulating tumor cells (CTCs) are tumor cells shed from solid tumor masses into the peripheral circulation and are key to tumor metastasis. Single‐cell sequencing has revealed that the genetic profiles of individual CTCs are highly heterogeneous and contribute to the poor outcome and prognosis of SCLC patients. Theoretically, phenotypic analysis of CTCs may be able to predict the diagnostic significance of new potential targets for metastatic tumors. In this paper, we will discuss in depth the heterogeneity of CTCs in SCLC and the value of CTCs for the diagnosis and prognosis of SCLC and as relevant tumor markers in metastatic SCLC. [ABSTRACT FROM AUTHOR]

Background: Detection rate and isolation yield of circulating tumor cells (CTCs) are low in lung cancer with approaches due to CTC invasiveness and heterogeneity. In this study, on the basis of the epithelial cell adhesion molecule (EpCAM) phenotype, markers of vimentin and epidermal growth factor receptor (EGFR) phenotype were added to jointly construct a precise and efficient CTC capture system for capture of lung cancer CTCs. Methods: A CTC capture system combined with EpCAM lipid magnetic bead (Ep-LMB)/vimentin lipid magnetic bead (Vi-LMB)/EGFR lipid magnetic bead (EG-LMB) was constructed, and its performance was tested. The amount of CTC captured in the blood of patients with lung cancer was detected by immunofluorescence identification and analyzed for clinical relevance. Results: The constructed CTC capture system has low cytotoxicity. The capture efficiency of lung cancer cells in phosphate belanced solution (PBS) system was 95.48%. The capture efficiency in the blood simulation system is 94.55%. The average number of CTCs in the blood of patients with lung cancer was 9.73/2 mL. The quantity distribution of CTCs is significantly correlated with tumor staging and metastasis. The area under the curve (AUC) of CTCs for the diagnosis of lung cancer was 0.9994 (95% CI = 0.9981-1.000, P <.0001). The cutoff value was 4.5/2 mL. The sensitivity was 99.39%, and the specificity was 96.88%. Conclusion: The EpCAM/vimentin/EGFR combined capture system has feasibility and high sensitivity in the detection of lung cancer CTC typing, which can be used as an auxiliary diagnostic indicator for lung cancer and is expected to promote the clinical application of CTCs. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) are noninvasive biomarkers that can indicate the therapeutic response and prognosis. The study aimed to investigate the cellular characteristics of CTCs focusing on monitoring during atezolizumab and bevacizumab (Atezo-Bev) therapy in patients with hepatocellular carcinoma (HCC). Peripheral blood samples were collected from 10 healthy controls and 40 patients with HCC. CTCs enriched using RosetteSep™ Human CD45 depletion cocktail were analyzed by multiparametric flow cytometry. CTC isolation was based on PanCK(+)CD45(−) cells, and CTCs exhibiting markers CD90, CD133, EpCAM, or vimentin. The total number of CTCs and the number of CTCs expressing CD90, CD133, EpCAM, and vimentin were correlated with the BCLC stage of HCC. The change in total CTC count accurately reflected the initial response to Atezo-Bev therapy. The numbers and mean fluorescence intensity of the CTC subsets expressing CD90 and EpCAM molecules decreased in patients with partial response/stable disease, and increased in patients with progressive disease and were markedly correlated with overall survival. CD90(+) and EpCAM(+) CTCs may be candidate biomarkers for the early prediction of the treatment response and the overall survival of patients with HCC receiving Atezo-Bev therapy.

Abstract Currently, tumor treatment modalities such as immunotherapy and targeted therapy have more stringent requirements for obtaining tumor growth information and require more accurate and easy‐to‐operate tumor information detection methods. Compared with traditional tissue biopsy, liquid biopsy is a novel, minimally invasive, real‐time detection tool for detecting information directly or indirectly released by tumors in human body fluids, which is more suitable for the requirements of new tumor treatment modalities. Liquid biopsy has not been widely used in clinical practice, and there are fewer reviews of related clinical applications. This review summarizes the clinical applications of liquid biopsy components (e.g., circulating tumor cells, circulating tumor DNA, extracellular vesicles, etc.) in tumorigenesis and progression. This includes the development process and detection techniques of liquid biopsies, early screening of tumors, tumor growth detection, and guiding therapeutic strategies (liquid biopsy‐based personalized medicine and prediction of treatment response). Finally, the current challenges and future directions for clinical applications of liquid biopsy are proposed. In sum, this review will inspire more researchers to use liquid biopsy technology to promote the realization of individualized therapy, improve the efficacy of tumor therapy, and provide better therapeutic options for tumor patients.

Introduction: Cancer is a leading cause of death worldwide and is a major cause of morbidity. To deal with this magnitude of cancers and their diagnostic and prognostics, a multitude of prognostic biomarkers for various cancers have been explored over the decades, with detection of circulating tumor cells (CTCs) in the peripheral blood being one of them. This study was undertaken to explore the routine procedure of cell block in the cytopathology lab to isolate and detect CTCs. Materials and Methods: In this cross-sectional study, 112 peripheral blood samples sent for routine blood investigations of various cancer patients were utilized for the preparation of cell block. The sections from the cell block were stained routinely and evaluated for the presence of CTCs. The statistical analysis was done using Mac Statplus software version 8.0. Results: The malignancies were tabulated as per the International Classification of Diseases for Oncology, third edition (ICD-O-3). The maximum number of cases were from C 50 (breast) - 41/112 (36.6%), followed by C15–C26 (Digestive organs) - 19/112 (16.9%), and C00–C14 (lip, oral cavity, and pharynx) - 18/112 (16.07%) cases. CTC was detected in six (5.35%) out of 112 cases, out of which three were from the breast and one each from category C6.9 (mouth), C32.0 (glottis), and C53.8 (cervix uteri). Conclusion: Among various advanced and molecular techniques available for the detection of CTCs, the cell block technique proves to be one of the effective methods, especially in resource-limited settings as these can further be utilized for additional diagnostic techniques similar to the ones employed for routine paraffin blocks.

Abstract Background The clinical relevance of circulating tumor cell-white blood cell (CTC-WBC) clusters in cancer prognosis is a subject of ongoing debate. This study aims to unravel their contentious predictive value for patient outcomes. Methods We conducted a comprehensive literature search of PubMed, Embase, and Cochrane Library up to December 2022. Eligible studies that reported survival outcomes and examined the presence of CTC-WBC clusters in solid tumor patients were included. Hazard ratios (HR) were pooled to assess the association between CTC-WBC clusters and overall survival (OS), as well as progression-free survival (PFS)/disease-free survival (DFS)/metastasis-free survival (MFS)/recurrence-free survival (RFS). Subgroup analyses were performed based on sampling time, treatment method, detection method, detection system, and cancer type. Results A total of 1471 patients from 10 studies were included in this meta-analysis. The presence of CTC-WBCs was assessed as a prognostic factor for overall survival and PFS/DFS/MFS/RFS. The pooled analysis demonstrated that the presence of CTC-WBC clusters was significantly associated with worse OS (HR = 2.44, 95% CI: 1.74–3.40, P

Detecting circulating tumor cells has exhibited great significance in treating cancers since its concentration is an index strongly associated with the development and transfer of the tumor. However, the present commercial method for CTC detection is still expensive, because special antibodies and complicated devices must be used for cell separation and imaging. Hence, it is quite necessary to apply alternative materials and methods to decrease the cost of CTC detection. In this article, we coated a cellulose acetate membrane with nanoparticles formed by the polymerization of melamine and furfural, creating a surface with nanoscale roughness for the highly efficient capture of the sparse CTCs in a blood sample. Subsequently, the CTCs on the surface can be quantitatively detected by colorimetry with the aid of a COF-based nanozyme. The detection limit (LOD) can be as low as 3 cells/mL, which is the lowest LOD among the colorimetric methods to our knowledge. Considering the low cost of fabricating the membrane for CTC capture and the robustness of nanozymes compared with natural enzymes, this CTC detection approach displays great potential to decrease the financial burden of commercial CTC detection.

Serum alpha-fetoprotein and radiologic imaging are the most commonly used tests for early diagnosis and dynamic monitoring of treatment response in hepatocellular carcinoma (HCC). However, the accuracy of these tests is limited, and they may not reflect the underlying biology of the tumor. Thus, developing highly accurate novel HCC biomarkers reflecting tumor biology is a clinically unmet need. Circulating tumor cells (CTCs) have long been proposed as a noninvasive biomarker in clinical oncology. Most CTC assays utilize immunoaffinity-based, size-based, and/or enrichment-free mechanisms followed by immunocytochemical staining to characterize CTCs. The prognostic value of HCC CTC enumeration has been extensively validated. Subsets of CTCs expressing mesenchymal markers are also reported to have clinical significance. In addition, researchers have been devoting their efforts to molecular characterizations of CTCs (e.g. genetics and transcriptomics) as molecular profiling can offer a more accurate readout and provide biological insights. As new molecular profiling techniques, such as digital polymerase chain reaction, are developed to detect minimal amounts of DNA/RNA, several research groups have established HCC CTC digital scoring systems to quantify clinically relevant gene panels. Given the versatility of CTCs to provide intact molecular and functional data that reflects the underlying tumor, CTCs have great potential as a noninvasive biomarker in HCC. Large-scale, prospective studies for HCC CTCs with a standardized protocol are necessary for successful clinical translation.

Background The MONARCH 2 trial (NCT02107703) showed the efficacy of abemaciclib, a cyclin-dependent kinase 4 & 6 inhibitor (CDK4/6i), in combination with fulvestrant for hormone receptor-positive, HER2-negative metastatic breast cancer (MBC). The aim of this analysis was to explore the prediction of circulating tumor cells (CTCs) stratification using machine learning for hypothesis generation of biomarker-driven clinical trials. Patients and Methods Predicted CTCs were computed in the MONARCH 2 trial through a K nearest neighbor (KNN) classifier trained on a dataset comprising 2436 patients with MBC. Patients were categorized into predicted Stage IVaggressive (pStage IVaggressive, ≥5 predicted CTCs) or predicted Stage IVindolent (pStage IVindolent, <5 predicted CTCs). Prognosis was tested in terms of progression-free-survival (PFS) and overall survival (OS) through Cox regression. Results Patients classified as predicted pStage IVaggressive and predicted pStage Stage IVindolent were, respectively, 183 (28%) and 461 (72%). After multivariable Cox regression, predicted CTCs were confirmed as independently associated with prognosis in terms of OS, together with ECOG performance status, liver involvement, bone-only disease, and treatment arm. Patients in the pStage Stage IVindolent subgroup treated with abemaciclib experienced the best prognosis both in terms of PFS and OS. The treatment effect of abemaciclib on OS was then explored through subgroup analysis, showing a consistent benefit across all subgroups. Conclusion This study is the first analysis of CTCs modeling for stage IV disease stratification. These results show the need to expand biomarker profiling in combination with CTCs stratification for improved biomarker-driven drug development. [ABSTRACT FROM AUTHOR]

Introduction: Cancer is a leading cause of death worldwide and is a major cause of morbidity. To deal with this magnitude of cancers and their diagnostic and prognostics, a multitude of prognostic biomarkers for various cancers have been explored over the decades, with detection of circulating tumor cells (CTCs) in the peripheral blood being one of them. This study was undertaken to explore the routine procedure of cell block in the cytopathology lab to isolate and detect CTCs. Materials and Methods: In this cross-sectional study, 112 peripheral blood samples sent for routine blood investigations of various cancer patients were utilized for the preparation of cell block. The sections from the cell block were stained routinely and evaluated for the presence of CTCs. The statistical analysis was done using Mac Statplus software version 8.0. Results: The malignancies were tabulated as per the International Classification of Diseases for Oncology, third edition (ICD-O-3). The maximum number of cases were from C 50 (breast) - 41/112 (36.6%), followed by C15–C26 (Digestive organs) - 19/112 (16.9%), and C00–C14 (lip, oral cavity, and pharynx) - 18/112 (16.07%) cases. CTC was detected in six (5.35%) out of 112 cases, out of which three were from the breast and one each from category C6.9 (mouth), C32.0 (glottis), and C53.8 (cervix uteri). Conclusion: Among various advanced and molecular techniques available for the detection of CTCs, the cell block technique proves to be one of the effective methods, especially in resource-limited settings as these can further be utilized for additional diagnostic techniques similar to the ones employed for routine paraffin blocks. [ABSTRACT FROM AUTHOR]

Background: The clinical relevance of circulating tumor cell-white blood cell (CTC-WBC) clusters in cancer prognosis is a subject of ongoing debate. This study aims to unravel their contentious predictive value for patient outcomes. Methods: We conducted a comprehensive literature search of PubMed, Embase, and Cochrane Library up to December 2022. Eligible studies that reported survival outcomes and examined the presence of CTC-WBC clusters in solid tumor patients were included. Hazard ratios (HR) were pooled to assess the association between CTC-WBC clusters and overall survival (OS), as well as progression-free survival (PFS)/disease-free survival (DFS)/metastasis-free survival (MFS)/recurrence-free survival (RFS). Subgroup analyses were performed based on sampling time, treatment method, detection method, detection system, and cancer type. Results: A total of 1471 patients from 10 studies were included in this meta-analysis. The presence of CTC-WBCs was assessed as a prognostic factor for overall survival and PFS/DFS/MFS/RFS. The pooled analysis demonstrated that the presence of CTC-WBC clusters was significantly associated with worse OS (HR = 2.44, 95% CI: 1.74–3.40, P < 0.001) and PFS/DFS/MFS/RFS (HR = 1.83, 95% CI: 1.49–2.24, P < 0.001). Subgroup analyses based on sampling time, treatment method, detection method, detection system, cancer type, and study type consistently supported these findings. Further analyses indicated that CTC-WBC clusters were associated with larger tumor size (OR = 2.65, 95% CI: 1.58–4.44, P < 0.001) and higher alpha-fetoprotein levels (OR = 2.52, 95% CI: 1.50–4.22, P < 0.001) in hepatocellular carcinoma. However, no significant association was found between CTC-WBC clusters and TNM stage, depth of tumor invasion, or lymph node metastasis in the overall analysis. Conclusions: CTC-WBC clusters are negative predictors for OS and PFS/DFS/MFS/RFS in patients with solid tumors. Monitoring CTC-WBC levels may provide valuable information for predicting disease progression and guiding treatment decisions. [ABSTRACT FROM AUTHOR]

To simply, quickly, and efficiently separate circulating tumor cells from blood has always been an enormous challenge. Leveraging the principle of inertial focusing, we here designed a simply structured microfluidic chip that maintained excellent CTC separation efficiency with high robustness and low velocity sensitivity across a broad velocity range. The parameter configuration of the chip was systematically examined, especially the most influential parameters such as the arc radius and arc angle. With optimal parameters, the designed chip achieved an outstanding particle separation efficiency of 99.8% and, more importantly, enabled the efficient separation and enrichment of CTCs in blood samples. This design can be readily integrated with other functional modules for further sample processing, serving as a promising tool for cancer diagnosis and therapeutics. [ABSTRACT FROM AUTHOR]

Background: Circulating tumor cell (CTC) counting may be a useful non-invasive biomarker that helps patients choose first-line treatment options. Nevertheless, the cost of CTC inspection may impose an economic burden on patients, necessitating the simultaneous consideration of both its clinical effectiveness and cost. We evaluated the cost-effectiveness of CTC count-guided chemotherapy and endocrine therapy as first-line therapy for HR+/HER2-metastatic breast cancer (MBC) from the perspective of US payers. Methods: Based on the STIC CTC trial, a Markov model was constructed for three health states, and health outcomes were measured in quality-adjusted life years (QALYs) and incremental cost-effectiveness ratio (ICER). One-way and probabilistic sensitivity analyses were performed to assess the robustness of the incremental cost per QALY. Results: The base-case analysis revealed that CTC count-driven treatment was associated with improved effectiveness by 0.07 QALYs and increased the overall cost by $9187.05 compared with clinician-driven first-line treatment choices, leading to an ICER of $138 354.15 per QALY. One-way sensitivity analysis indicated that the model was most sensitive to the cost of treatment for neutropenia and the utility for PFS; probability sensitivity analysis indicated that CTC count-driven treatment choices would be considered the cost-effective option at a willingness-to-pay threshold of $150 000 per QALY. Conclusions: The findings of this cost-effectiveness analysis suggest that, at the current price of CTC enumeration, choosing first-line treatment options based on CTC count is a cost-effectiveness approach for treating patients with HR+/HER2- MBC in the US.

Circulating tumor cells (CTCs) that undergo epithelial-to-mesenchymal transition (EMT) can provide valuable information regarding metastasis and potential therapies. However, current studies on the EMT overlook alternative splicing. Here, we used single-cell full-length transcriptome data and mRNA sequencing of CTCs to identify stage-specific alternative splicing of partial EMT and mesenchymal states during pancreatic cancer metastasis. We classified definitive tumor and normal epithelial cells via genetic aberrations and demonstrated dynamic changes in the epithelial-mesenchymal continuum in both epithelial cancer cells and CTCs. We provide the landscape of alternative splicing in CTCs at different stages of EMT, uncovering cell-type-specific splicing patterns and splicing events in cell surface proteins suitable for therapies. We show that MBNL1 governs cell fate through alternative splicing independently of changes in gene expression and affects the splicing pattern during EMT. We found a high frequency of events that contained multiple premature termination codons and were enriched with C and G nucleotides in close proximity, which influence the likelihood of stop codon readthrough and expand the range of potential therapeutic targets. Our study provides insights into the EMT transcriptome’s dynamic changes and identifies potential diagnostic and therapeutic targets in pancreatic cancer.

Objective To investigate the detection and clinical application studies based on malaria protein VAR2CSA(VAR2) synthetic peptide specifically binding to circulating tumor cells (CTCs). Methods The peptide sequence of the functional structural domain of malaria protein VAR2CSA was selected to synthesize VAR2 that enriched CTCs based on nano-microfluidic technology, and used VAR2 synthetic peptide to detect enriched cells to establish a novel detection protocol for CTCs. Cellular immuno-fluorescence staining, flow cytometry and tumor cell doping recovery assays were used to analyze the specific binding and recovery efficiency of VAR2 synthetic peptide with tumor cells of different tissue sources and compare with immuno-staining-fluorescence in situ hybridization (imFISH) assay. Twenty-two colorectal cancer patients and twenty-two healthy individuals were selected, and the VAR2 synthetic peptide was used to detect peripheral blood CTCs in both. The VAR2 synthetic peptide was used to compare the positive detection rate of CTCs in patients with different TNM stages. Results The VAR2 synthetic peptide bound specifically to tumor cells collected from different tissue sources of human colon cancer cell line SW620, human breast cancer cell line MCF7 and human esophageal squamous carcinoma cell line KYSE180.The VAR2 synthetic peptide detected 100 SW620, MCF7 and KYSE180 tumor cells mixed into 3 mL of peripheral blood with recovery rates of 82.8%±8.41%, 56.2%±3.57%, 86.6%±8.19%, which were not significantly different from the imFISH assay. The VAR2 synthetic peptide assay recoveries of 50, 100, and 200 SW620 tumor cells were 80.7%, 82.8%, and 84.7%, respectively, which did not correlate significantly with the number of adulterated tumor cells. The VAR2 synthetic peptide detected 63.6% positive CTCs in peripheral blood samples from patients with clinical colorectal cancer, which was higher than the 0.0% positive CTCs rate in the group of healthy subjects, and also correlated with depth of invasion, lymph node metastasis, distant metastasis, and clinical stage (P＜0.05). Conclusions The VAR2-based synthetic peptide may be applied in specific and non-antibody dependent CTCs detection in different tissue samples and so has good clinical application value for clinical staging and severity evaluation of tumor patients.

Abstract In recent decades, using circulating tumor cell (CTC), circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA), exosomes and etc. as liquid biomarkers has received enormous attention in various tumors, including breast cancer (BC). To date, efforts in the area of liquid biopsy predominantly focus on the analysis of blood-based markers. It is worth noting that the identifications of markers from non-blood sources provide unique advantages beyond the blood and these alternative sources may be of great significance in offering supplementary information in certain settings. Here, we outline the latest advances in the analysis of non-blood biomarkers, predominantly including urine, saliva, cerebrospinal fluid, pleural fluid, stool and etc. The unique advantages of such testings, their current limitations and the appropriate use of non-blood assays and blood assays in different settings are further discussed. Finally, we propose to highlight the challenges of these alternative assays from basic to clinical implementation and explore the areas where more investigations are warranted to elucidate its potential utility.

Abstract Background Systemic therapy is the primary treatment for advanced thymic malignancies. However, there is an urgent need to improve clinical outcome. Personalized treatment based on predictive biomarkers is a potential approach to address this requirement. In this study, we aimed to show the correlation between drug sensitivity tests on CTCs‐derived organoids and clinical response in patients with thymic malignancies. This approach carries the potential to create personalized cancer avatars and improve treatment outcome for patients. Methods We previously reported potential treatment outcome prediction with patient‐derived organoids (cancer avatars) in patients with pancreatic ductal adenocarcinoma. To further investigate the feasibility of this approach in advanced thymic malignancies, we conducted a study in which 12 patients were enrolled and 21 liquid biopsies were performed. Results Cancer avatars were successfully derived in 16 out of 21 samples (success rate 76.2%). We found a sensitivity of 1.0 and specificity of 0.6 for drug sensitivity tests on the cancer avatars, and a two‐tailed Fisher's exact test revealed a significant correlation between drug sensitivity tests and clinical responses (p = 0.0275). Conclusion This study supports the potential of circulating tumor cell‐derived organoids to inform personalized treatment for advanced thymic malignancies. Further validation of this proof of concept finding is ongoing.

The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) is the standard-of-care for estrogen receptor (ER)-positive, HER2-negative (ER+/HER2− advanced/metastatic breast cancer (mBC). However, the impact of CDK4/6i on circulating immune cells and circulating tumor cells (CTCs) in patients receiving CDK4/6i and ET (CDK4/6i+ET) remains poorly understood. This was a prospective cohort study including 44 patients with ER+/HER2− mBC treated with CDK4/6i+ET in either first or second line. Peripheral blood samples were collected before (baseline) and 3 months (t2) after therapy. Immune cell’s subsets were quantified by flow cytometry, and microfluidic-captured CTCs were counted and classified according to the expression of cytokeratin and/or vimentin. Patients were categorized according to response as responders (progression-free survival [PFS] ≥ 6.0 months; 79.1%) and non-responders (PFS < 6.0 months; 20.9%). CDK4/6i+ET resulted in significant changes in the hematological parameters, including decreased hemoglobin levels and increased mean corpuscular volume, as well as reductions in neutrophil, eosinophil, and basophil counts. Specific immune cell subsets, such as early-stage myeloid-derived suppressor cells, central memory CD4+ T cells, and Vδ2+ T cells expressing NKG2D, decreased 3 months after CDK4/6i+ET. Additionally, correlations between the presence of CTCs and immune cell populations were observed, highlighting the interplay between immune dysfunction and tumor dissemination. This study provides insights into the immunomodulatory effects of CDK4/6i+ET, underscoring the importance of considering immune dynamics in the management of ER+/HER2− mBC.

A major barrier to the emergence of distant metastases is the survival of circulating tumor cells (CTCs) within the vasculature. Lethal stressors, including shear forces from blood flow, anoikis arising from cellular detachment, and exposure to natural killer cells, combine to subvert the ability of primary tumor cells to survive and ultimately seed distant lesions. Further attenuation of this rate-limiting process via therapeutic intervention offers a very attractive opportunity for improving cancer patient outcomes, in turn prompting the need for a deeper understanding of the molecular and cellular mechanisms underlying CTC viability. MUC4 is a very large and heavily glycosylated protein expressed at the apical surfaces of the epithelia of a variety of tissues, is involved in cellular growth signaling and adhesiveness, and contributes to the protection and lubrication of cellular linings. Analysis of patient-matched breast tumor specimens has demonstrated that MUC4 protein levels are upregulated in metastatic lesions relative to primary tumor among all breast tumor subtypes, pointing to a possible selective advantage for MUC4 overexpression in metastasis. Analysis of a genetically engineered mouse model of HER2-positive breast cancer has demonstrated that metastatic efficiency is markedly suppressed with Muc4 deletion and Muc4-knockout tumor cells are poorly associated with platelets and white blood cells known to support CTC viability. In this review, we discuss the diverse roles of MUC4 in tumor progression and metastasis and propose that intervening in MUC4 intercellular interactions with binding partners on blood-borne aggregating cells could potentially thwart breast cancer metastatic efficiency.

Malignant tumors seriously threat the health of the Chinese population. Circulating tumor cell (CTC), as molecular markers in liquid biopsy, have the advantage of being non-invasive and longitudinal monitoring of tumor progression and efficacy. In vitro CTC enrichment detection methods are divided into two types: based on molecular characteristics and based on physical characteristics. In recent years, a series of comprehensive CTC enrichment methods targeting different molecular characteristics have been developed, as well as the use of subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH) for enrichment detection of gastric cancer CTC. Microfluidic technology can separate and enrich CTC on the same chip based on their molecular and physical characteristics simultaneously, helping to achieve more efficient and sensitive CTC enrichment detection. Compared with the in vitro CTC enrichment platform, the in vivo CTC enrichment platform has higher capture efficiency and detection sensitivity, but it is an invasive examination, and the impact on patients still needs further study. High CTC level is closely related to the disease control rate after gastric cancer chemotherapy and is an independent predictor of poor prognosis in gastric cancer patients. Among non-metastatic malignancies, increased CTC counts after surgery in patients with esophageal cancer and colorectal cancer are strongly associated with worse overall survival. Since CTC are highly heterogeneous, changes in CTC can be longitudinally monitored to provide a reference for the treatment of malignant tumors. Research on gastric cancer has shown that the positivity rate, size of circulating tumor microemboli (CTM) and gene expression level of CTC are closely related to the ability of invasion, metastasis, drug resistance and prognosis. The dynamic metastasis process and pattern of CTC play an important role in tumor metastasis. For the detection of CTC, it is necessary to establish unified standards and verification methods, and develop multi-omics research technology.

Liver cancer is currently the third leading cause of cancer-related mortality worldwide. Due to late diagnosis and difficulty in monitoring, there is a pressing need for early detection and recurrence monitoring in patients with liver cancer. Recent advancements in liquid biopsy technology, like circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes, have made it possible to obtain a tumor’s characteristics and dynamic monitor easily. This, in turn, helps in identifying a personalized therapy for individual patients. However, the application progress of liquid biopsy techniques in liver cancer lag behind due to various challenges in clinical practice. In this review, we aim to provide insights into the development of liquid biopsy technology in liver cancer, highlighting its clinical significance in diagnosis, prognosis and treatment response prediction. We hope to focus on the key opportunities and challenges associated with these biomarkers and inspire a potential direction for future research.

In recent decades, using circulating tumor cell (CTC), circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA), exosomes and etc. as liquid biomarkers has received enormous attention in various tumors, including breast cancer (BC). To date, efforts in the area of liquid biopsy predominantly focus on the analysis of blood-based markers. It is worth noting that the identifications of markers from non-blood sources provide unique advantages beyond the blood and these alternative sources may be of great significance in offering supplementary information in certain settings. Here, we outline the latest advances in the analysis of non-blood biomarkers, predominantly including urine, saliva, cerebrospinal fluid, pleural fluid, stool and etc. The unique advantages of such testings, their current limitations and the appropriate use of non-blood assays and blood assays in different settings are further discussed. Finally, we propose to highlight the challenges of these alternative assays from basic to clinical implementation and explore the areas where more investigations are warranted to elucidate its potential utility. [ABSTRACT FROM AUTHOR]

Purpose: Function of survivin protein (encoded by BIRC5) in circulating tumor cells (CTCs) of osteosarcoma (OS) has not been investigated. The goal of this study is to determine whether the expression of survivin protein of CTCs is associated with circulating immune cell infiltration and disease prognosis of OS. Methods: Blood samples of 20 patients with OS were collected. CanPatrol™ CTC enrichment technology combined with in situ hybridization (ISH) was applied to enrich and test CTCs and survivin protein. Bioinformation analysis combined with data of routine blood test was used to verify the association between survivin and immune cell infiltration in circulatory system. To screen independent prognostic factors, Kaplan–Meier survival curve, univariate and multivariable Cox regression analyses were performed. Results: Bioinformatics analysis showed that BIRC5 was strongly negatively related to lymphocyte, including T cell, NK cell and B cell, which released that BIRC5 played a key role in immune escape via reducing immune cell infiltration in circulatory system. Meanwhile, the number of survivin+ CTCs was significantly negatively connection with lymphocyte count (R = −0.56, p = 0.011), which was consistent with bioinformatics analysis. Kaplan–Meier curve showed that the overall survival rate in high survivin+ CTCs group was significantly lower than low group (88.9% vs 36.4%, p = 0.04). Multivariable Cox regression analyses showed that survivin+ CTCs were an independent prognostic factor (p = 0.019). Conclusion: These findings suggested that survivin protein played a key role in immune escape of CTCs and the presence of survivin+ CTCs might be a promising prognostic factor in OS patients. [ABSTRACT FROM AUTHOR]

Objective To investigate the detection and clinical application studies based on malaria protein VAR2CSA (VAR2) synthetic peptide specifically binding to circulating tumor cells (CTCs). Methods The peptide sequence of the functional structural domain of malaria protein VAR2CSA was selected to synthesize VAR2 that enriched CTCs based on nano-microfluidic technology, and used VAR2 synthetic peptide to detect enriched cells to establish a novel detection protocol for CTCs. Cellular immuno-fluorescence staining, flow cytometry and tumor cell doping recovery assays were used to analyze the specific binding and recovery efficiency of VAR2 synthetic peptide with tumor cells of different tissue sources and compare with immuno-staining-fluorescence in situ hybridization (imFISH) assay. Twenty-two colorectal cancer patients and twenty-two healthy individuals were selected, and the VAR2 synthetic peptide was used to detect peripheral blood CTCs in both. The VAR2 synthetic peptide was used to compare the positive detection rate of CTCs in patients with different TNM stages. Results The VAR2 synthetic peptide bound specifically to tumor cells collected from different tissue sources of human colon cancer cell line SW620, human breast cancer cell line MCF7 and human esophageal squamous carcinoma cell line KYSE180.The VAR2 synthetic peptide detected 100 SW620, MCF7 and KYSE180 tumor cells mixed into 3 mL of peripheral blood with recovery rates of 82.8%±8.41%, 56.2%±3.57%, 86.6%±8.19%, which were not significantly different from the imFISH assay. The VAR2 synthetic peptide assay recoveries of 50, 100, and 200 SW620 tumor cells were 80.7%, 82.8%, and 84.7%, respectively, which did not correlate significantly with the number of adulterated tumor cells. The VAR2 synthetic peptide detected 63.6% positive CTCs in peripheral blood samples from patients with clinical colorectal cancer, which was higher than the 0.0% positive CTCs rate in the group of healthy subjects, and also correlated with depth of invasion, lymph node metastasis, distant metastasis, and clinical stage (P＜0.05). Conclusions The VAR2-based synthetic peptide may be applied in specific and non-antibody dependent CTCs detection in different tissue samples and so has good clinical application value for clinical staging and severity evaluation of tumor patients. [ABSTRACT FROM AUTHOR]

The application of immunomagnetic nanoparticles (IMNs) enables the isolation of rare circulating tumor cells (CTCs) from blood. Precise capture of CTCs with specific tumor phenotypes is achieved by attaching specific ligands to the surface of IMNs. The captured CTCs need to exhibit high bioactivity and proliferation ability for subsequent detection after co-culturing. The process of CTC enrichment using magnetic nanoparticles entails several issues, such as nonspecific capture and biological toxicity. Modifying the size of nanoparticles allows for altering the capture efficiency of nanoparticles for CTCs, as well as their biological activity and proliferation ability. In this study, two different sizes of magnetic nanoparticles are utilized to capture CTCs under diverse conditions. The capture efficiency of magnetic nanoparticles is assessed at different incubation times, along with determining the time needed to achieve and sustain the optimal capture efficiency. Fluorescence staining is employed to detect the bioactivity and cellular integrity of the captured CTCs. Subsequent to co-culturing with nanoparticles, the number of CTCs is measured every 24 h, and the survival rate is determined after 96 h. [ABSTRACT FROM AUTHOR]

Despite increasing interest in the study of circulating tumor cells (CTC) subsets, especially epithelial-mesenchymal transition (EMT) and stem cells subsets of CTC that play a key role in tumor recurrence and metastasis, there is no evidence from meta-analyses that shows the correlation between stem-like CTCs and prognosis in cancer patients. Thus, we performed a meta-analysis to assess its prognostic value. Sixteen articles were screened by searching the PubMed, Embase, Cochrane, China National Knowledge Internet (CNKI) and Wanfang databases. The hazard ratio (HR) and 95% confidence interval (95% CI) extracted from each article were summarized. Patients with positive stem-like CTCs in peripheral blood had significantly shorter overall survival (OS, HR: 2.58, 95% CI 1.76–3.79, P < 0.00001), progression-free survival (PFS, HR: 2.21, 95% CI 1.26–3.89, P = 0.006) and disease-free survival (DFS, HR: 2.53, 95% CI: 1.12–5.70, P = 0.03). This study provides the first meta-analysis evidence for the prognostic value of stem-like CTCs, demonstrating that these cells are associated with poor prognosis in cancer patients. Systematic review registration CRD42022322062 [ABSTRACT FROM AUTHOR]

Purpose: Prostate cancer (PCa) is one of the most common malignancies in men and one of the leading causes of cancer-related deaths; circulating tumor cells (CTC) are malignant cells that have broken off from original tumor or metastatic sites and extravasated into the blood vessels either naturally or maybe as a consequence of surgical procedures. This study aims to explore the feasibility of liquid biopsy technique to diagnose prostate cancer. Method: We constructed an assay platform integrating magnetic separation and fluorescence in situ hybridization (FISH) to effectively capture prostate cancer CTCs and evaluate the distribution between healthy volunteers and prostate cancer patients, respectively. Results: There was a significant difference in the number of CTCs between the healthy population and prostate cancer patients (P < 0.001). The results of the study showed that the CTCs capture identification system has good sensitivity and specificity in identifying prostate cancer patients. Conclusion: The CTCs test allows us to accurately identify patients who are at high risk for prostate cancer, allowing for early intervention and treating patients effectively. [ABSTRACT FROM AUTHOR]