Liquid biopsies provide a less-invasive option to tissue biopsies for the early diagnosis, prognosis, and tailored therapy of colorectal cancer (CRC). CRC is a major cause of cancer-related death, and early identification is essential for improving patient outcomes.

Conventional diagnostic techniques, including colonoscopy and tissue biopsy, may be enhanced by liquid biopsies that examine circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), extracellular vesicles (EVs), and other indicators present in body fluids. These markers provide significant insights into tumor biology, heterogeneity, and therapeutic response. CTCs detected in early-stage CRC have prognostic significance for disease recurrence and survival, while ctDNA investigation may uncover genetic mutations, epigenetic alterations, and tumor development. The identification of ctDNA in minimal residual disease (MRD) postsurgery correlates with an elevated risk of recurrence and unfavorable prognosis, underscoring its use in assessing treatment effectiveness. Furthermore, non-coding RNAs (ncRNAs) contained inside EVs provide potential prospective biomarkers and therapeutic targets, facilitating diagnosis and treatment assessment. Notwithstanding the potential of liquid biopsies, obstacles persist in assay standardization, sensitivity enhancement, and the management of tumor heterogeneity. Additional extensive research is required to determine their function in clinical practice.

Overall, liquid biopsies serve as a potential instrument for real-time monitoring, evaluating therapy responses, and directing individualized therapeutic strategies in CRC patients.

Detecting circulating tumor cells (CTCs) is challenging due to their low presence and heterogeneity. Traditional methods using EpCAM-based separation struggle with CTCs that have undergone epithelial-mesenchymal transition, as this results in lower EpCAM expression. This study presents the use of silica-coated magnetic nanobeads functionalized with streptavidin for CTC capture. Using the FETCH magnetic separation system, we validated the capture efficiency of our beads on tumor cells with varying EpCAM expression. Our beads showed superior capture rates for LNCaP (97%), PC3-9 (91%), PC3 (23%), A549 (22%), and T24 (8%) cells compared to commercial MojoSort™ beads. Despite slightly higher nonspecific binding than CellSearch, our beads demonstrated improved sensitivity for EpCAMlow cells, suggesting they have promise for enhanced CTC capture.

In recent years, liquid biopsy has gained prominence as an emerging biomarker in cancer research, providing critical insights into tumor biology and metastasis. Technological advancements have enabled its integration into clinical practice, with ongoing trials demonstrating encouraging outcomes. Key applications of liquid biopsy include early cancer detection, cancer staging, prognosis evaluation, and real-time monitoring of tumor progression to optimize treatment decisions. In this review, we present a comprehensive conceptual framework for liquid biopsy, discuss the challenges in its research and clinical application, and highlight its significant potential in identifying therapeutic targets and resistance mechanisms across various cancer types. Furthermore, we explore the emerging role of liquid biopsy-based multicancer screening, which has shown promising advancements. Looking ahead, standardization, multi-omics coanalysis, and the advancement of precision medicine and personalized treatments are expected to drive the future development and integration of liquid biopsy into routine clinical workflows, enhancing cancer diagnosis and treatment management.

In the era of precision medicine, mounting evidence suggests that the time of therapy administration, or chronotherapy, has a great impact on treatment outcomes. Chronotherapy involves planning treatment timing by considering circadian rhythms, which are 24 h oscillations in behavior and physiology driven by synchronized molecular clocks throughout the body. The value of chronotherapy in cancer treatment is currently under investigation, notably in the effects of treatment timing on efficacy and side effects. Immune checkpoint inhibitor (ICI) therapy is a promising cancer treatment. However, many patients still experience disease progression or need to stop the therapy early due to side effects. There is accumulating evidence that the time of day at which ICI therapy is administered can have a substantial effect on ICI efficacy. Thus, it is important to investigate the intersections of circadian rhythms, chronotherapy, and ICI efficacy. In this review, we provide a brief overview of circadian rhythms in the context of immunity and cancer. Additionally, we outline current applications of chronotherapy for cancer treatment. We synthesize the 29 studies conducted to date that examine the impact of time-of-day administration on the efficacy of ICI therapy, its associated side effects, and sex differences in both efficacy and side effects. We also discuss potential mechanisms underlying these observed results. Finally, we highlight the challenges in this area and future directions for research, including the potential for a chronotherapeutic personalized medicine approach that tailors the time of ICI administration to individual patients' circadian rhythms.

Circulating tumor cells (CTCs) are key indicators of tumor metastasis. Effective clearance of CTCs can reduce the probability of metastasis. We designed a system for the real-time dynamic monitoring and clearance of CTCs, capable of monitoring and clearing CTCs in the living circulatory system. Experimental results showed that pulsed laser treatment significantly affects the clearance of melanoma CTCs. Through in vivo imaging of small animals and survival analysis of mice, we observed that CTC clearance could reduce the size of distant metastatic lesions and prolong the lifespan of the mice. Additionally, we set up a hemolysis experiment to demonstrate that the laser energy used does not cause damage to red blood cells. This study is based on the physical and mechanical destruction of CTCs, meaning there is no issue of drug resistance. This provides a novel approach and technical means for suppressing tumor metastasis and extending the lifespan of patients in clinical settings.

Isolation and characterization of circulating tumor cells (CTCs) present a noninvasive alternative to monitor disease progression in individual patients. However, the heterogeneous lineage specificity of CTCs makes it difficult to isolate and identify possible CTCs by a liquid biopsy. Better label-free methods for the isolation of viable CTCs are needed. Our solution is a combined approach that is inherently epitope independent. Cells are separated by size-sensitive acoustophoresis using an ultrasonic standing wave field, followed by size-insensitive, acoustic barrier-medium focusing, which enables the enrichment of viable cancer cells in blood. With standard acoustophoresis in homogeneous medium, lymphocytes and monocytes were efficiently removed, while removal of granulocytes from the target MCF7 breast cancer cells was not possible due to overlapping acoustic migration velocities for viable cells. Remaining granulocytes were removed by a second separation step with an acoustic impedance barrier-medium selectively blocking the transport of MCF7 cells to generate a clean cancer cell fraction. For two series of 500 mL samples containing 5 × 105 white blood cells, spiked with 2 × 104 or 1 × 103 MCF7 cells, the recovery of MCF7 cells was 77.3% with a 99.9% depletion of white blood cells in the final cancer cell fraction. The most abundant contaminating cell type was granulocytes (85.9% of remaining cells). Nearly all lymphocytes (99.996%) and monocytes (99.995%) were depleted. A two-step acoustic cell separation based on cell size and acoustic impedance is well suited to generate a purified cancer cell fraction as a preparatory step for downstream single-cell analysis.

Circulating tumor cells (CTCs) are rare (a few cells per milliliter of blood) and mostly isolated as single-cell CTCs (scCTCs). CTC clusters (cCTCs), even rarer, are of growing interest, notably because of their higher metastatic potential, but very difficult to isolate.

We introduce gravity-based microfiltration (GµF) for facile isolation of cCTCs using in-house fabricated microfilters and 3D printed cartridges. Optimal flow rate and pore size for cCTC isolation are determined by GµF of cultured ovarian single cells and cell clusters spiked in healthy blood. We perform GµF of blood from orthotopic ovarian cancer mouse models and characterize the morphological features of scCTCs and cCTCs, and the expression of molecular markers for aggressiveness. Finally, we analyze blood from 17 epithelial ovarian cancer patients with either localized or metastatic disease, and from 13 colorectal cancer liver metastasis patients.

Here, we show that GµF optimized for cell cluster isolation captures cCTCs from blood while minimizing unwanted cluster disaggregation, with ~85% capture efficiency. We detect cCTCs in every patient, with between 2-100+ cells. We find cCTCs represent between 5-30% of all CTC capture events, and 10-80% of the CTCs are clustered; remarkably, in 10 patients, most CTCs are circulating not as scCTCs, but as cCTCs.

GµF uncovers the unexpected prevalence and frequency of cCTCs including sometimes very large ones in epithelial ovarian cancer patients, and motivates additional studies to uncover their properties and role in disease progression.

Metastasis is the leading cause of death in tumor patients, with circulating tumor cells (CTCs) serving as key biomarkers for tumor progression, metastasis, and recurrence. CTC quantity is closely linked to tumor dynamics, which are influenced by biological rhythms. Studying CTC distribution under various physiological conditions provides insights into metastasis mechanisms. However, due to the low abundance of CTCs, detection accuracy is limited, especially with small blood samples, making continuous data collection challenging. To address this, we developed a dual-channel miniaturized in vivo fluorescence microscopy system for real-time monitoring of CTCs in experimental animals. This system, which can be fixed to the head or back, enables dynamic, quantitative analysis of CTCs in the circulatory system. It offers a valuable tool for investigating tumor metastasis rhythms, drug evaluation, and prognostic assessment in freely moving animals, advancing research in metastasis mechanisms and cancer treatment.

The existence of cancer stem cells (CSCs) in various tumors has become increasingly clear in addition to their prominent role in therapy resistance, metastasis, and recurrence. For early diagnosis, disease progression monitoring, and targeting, there is a high demand for clinical-grade methods for quantitative measurement of CSCs from patient samples. Despite years of active research, standard measurement of CSCs has not yet reached clinical settings, especially in the case of solid tumors. This is because detecting this plastic heterogeneous population of cells is not straightforward. This review summarizes various techniques, highlighting their benefits and limitations in detecting CSCs from patient samples. In addition, methods designed to detect CSCs based on secreted and niche-associated signaling factors are reviewed. Spatial and single-cell methods for analyzing patient tumor tissues and noninvasive techniques such as liquid biopsy and in vivo imaging are discussed. Additionally, methods recently established in laboratories, preclinical studies, and clinical assays are covered. Finally, we discuss the characteristics of an ideal method as we look toward the future.

Circulating tumor cells (CTCs) have attracted significant interest as a biomarker for cancer diagnosis. In this study, we judiciously constructed a recombinant MUC1-dependent adenovirus (rAdF35-MUC1) that can selectively replicate and overexpress copepod super green fluorescent proteins (copGFP) in MUC1-positive tumor cells to investigate its role in the detection of CTCs.

We conducted a comparative study between rAdF35-MUC1 and the existing hTERT-dependent adenovirus (rAdF35-hTERT). Breast cancer cell lines and healthy human peripheral blood mononuclear cells (PBMCs) were infected with both viral constructs to evaluate infection efficiency and the incidence of false-positive cells. CTC Model Samples were employed to determine detection rates, and clinical samples from breast cancer patients were analyzed to preliminarily evaluate the efficacy of CTC detection in a clinical context.

In preclinical and clinical studies, rAdF35-MUC1 exhibited a significantly high detection efficiency for breast cancer cells, outperforming the existing hTERT-dependent adenovirus (rAdF35-hTERT), especially in detecting CTCs at low quantities. Moreover, rAdF35-MUC1 demonstrated reduced incidence of false positives in healthy PBMCs compared to rAdF35-hTERT.

In brief, rAdF35-MUC1 emerges as a potent tool for the sensitive and specific identification of CTCs derived from breast cancer patients, holding clinical translation potential for advancing cancer (early) diagnosis, treatment monitoring, and prognosis.

Circulating tumour cells (CTCs) and tumour-derived extracellular vesicles (tdEVs) have great potential for monitoring therapy response and early detection of tumour relapse, facilitating personalized adjuvant therapeutic strategies. However, their low abundance in peripheral blood limits their informative value. In this study, we explored the presence of CTCs and tdEVs collected intraoperatively from a tumour-draining vein (DV) and via a central venous catheter (CVC) prior to tumour resection.

CellSearch analyses of 395 blood samples from 306 patients with gastrointestinal tumours and 93 blood samples from healthy donors were used to establish and validate gates for the automated detection of CTCs and tdEVs with ACCEPT software and R scripts. The selected gate settings were applied to 227 samples of 142 patients with colorectal cancer (CRC) from two independent collectives. Phenotypic features were obtained via numeric analysis of their fluorescence signals (e.g. size, shape, and intensity) and were used for calculating diversity using Shannon index (SI) of clusters generated via the k-means algorithm after Uniform Manifold Approximation and Projection (UMAP) pre-processing, and standard deviation (SD).

CTCs and tdEVs were more abundant in the DV samples compared to CVC samples (p < 0.05). tdEVs were detected in higher numbers than CTCs in both compartments. Importantly, tdEVs in CVCs were associated with tumor spread, whereas CTCs in DVs were linked to tumor size. In both compartments, the prognostic value of tdEVs for overall survival (OS) surpassed that of CTCs, as demonstrated by univariate, multivariate, and Kaplan-Meier analyses. CTCs and tdEVs in DVs were phenotypically distinct, being larger, more eccentric, and displaying stronger cytokeratin intensities (p < 0.05) compared to those in CVC samples. Furthermore, increased diversity in CTC and tdEV phenotypes was significantly associated with shorter survival, validating the prognostic relevance of the SD-diversity metric.

Our study demonstrates that DV sampling significantly enhances the detection of prognostically relevant CTCs and tdEVs in CRC patients, underscoring the superior prognostic significance of tdEVs compared to CTCs. Importantly, the combined phenotypic diversity of both markers emerges as a more powerful biomarker than their enumeration alone. These findings suggest that comprehensive, automated analysis of CTCs and tdEVs in DVs may open new avenues for tailoring individualized therapies in CRC patients.

Metastasis in patients with oral squamous cell carcinoma has been associated with a poor prognosis. However, sensitive and reliable tests for monitoring their occurrence are unavailable, with the exception of PET-CT. Circulating tumor cells and cell-free DNA have emerged as promising biomarkers for determining treatment efficacy and as prognostic predictors in solid tumors such as breast cancer and colorectal cancer. Hence, this study aimed to determine the potential role of liquid biopsy, circulating tumor cells, and cell-free DNA as biomarkers of oral squamous cell carcinoma. Thirteen patients with primary oral squamous cell carcinoma who visited our hospital between 2022 and 2023 were recruited, and plasma samples were collected from each patient preoperatively and postoperatively. We examined the relationship between the prognosis, the number of circulating tumor cells per four milliliters of peripheral blood, and the amount of cell-free DNA per milliliter of serum or the gene mutation in cell-free DNA. We observed no correlation between the number of preoperative circulating tumor cells and metastatic events. However, the number of circulating tumor cell clusters or the amount of preoperative cell-free DNA in metastatic cases was higher than that in non-metastatic cases. In oral squamous cell carcinoma, circulating tumor cell clusters or cell-free DNA levels may help inform management decisions regarding metastasis. However, further studies are required to provide a possible window for therapeutic interventions.

Cancer has a high mortality rate across the globe, and tissue biopsy remains the gold standard for tumor diagnosis due to its high level of laboratory standardization, good consistency of results, relatively stable samples, and high accuracy of results. However, there are still many limitations and drawbacks in the application of tissue biopsy in tumor. The emergence of liquid biopsy provides new ideas for early diagnosis and prognosis of tumor. Compared with tissue biopsy, liquid biopsy has many advantages in the diagnosis and treatment of various types of cancer, including non-invasive, quickly and so on. Currently, the application of liquid biopsy in tumor detection has received widely attention. It is now undergoing rapid progress, and it holds significant potential for future applications. Around now, liquid biopsies encompass several components such as circulating tumor cells, circulating tumor DNA, exosomes, microRNA, circulating RNA, tumor platelets, and tumor endothelial cells. In addition, advances in the identification of liquid biopsy indicators have significantly enhanced the possibility of utilizing liquid biopsies in clinical settings. In this review, we will discuss the application, advantages and challenges of liquid biopsy in some common tumors from the perspective of diverse systems of tumors, and look forward to its future development prospects in the field of cancer diagnosis and treatment.

Circulating tumor cells (CTCs) are pivotal in the distant metastasis of tumors, serving as one of the primary materials for liquid biopsy. They hold significant clinical importance in assessing prognosis, predicting efficacy, evaluating therapeutic outcomes, and studying recurrence, metastasis, and resistance mechanisms in cancer patients. Nevertheless, the rareness and heterogeneity of CTC and the complexity of metastasis make the clinical application of CTC detection confront many challenges, which may need to be settled by some practical strategies. This article will review the content mentioned above.

A fundamental prerequisite for the development of clinically manifested metastases in uveal melanoma (UM) is the hematogenous dissemination of melanoma cells, which circulate in the systemic circulation, deposit in organs and can subsequently colonize the organs.

This article presents a review of the literature on the detection of circulating tumor cells (CTC) from UM and their relevance for the clinical and pathophysiological aspects of the malignancy.

Since the first description of CTC in UM patients 20 years ago, only 20 articles have been published on the detection of intact CTCs in UM. Besides demonstrating the presence of CTCs in the systemic circulation, these articles give insights into the pathophysiology and chronology of CTC dissemination, the diagnostics and risk stratification of UM.

The detection of CTC in UM has proved to be beneficial in many ways. The early time of cell dissemination and also the associated factors should lead to a rethinking in the clinical routine in the context of the fatal danger of metastases.

Cancer vaccines have garnered attention as a potential treatment for cancer metastases. Nevertheless, the clinical response rate to vaccines remains < 30%. Nanoparticles stabilize vaccines and improve antigen recognition and presentation, resulting in high tumor penetration or accumulation, effective co-distribution of drugs to the secondary lymphatic system, and adaptable antigen or adjuvant administration. Such vaccine-like nanomedicines have the ability to eradicate the primary tumors as well as to prevent or eliminate metastases. This review examines state-of-the-art nanocarriers developed to deliver tumor vaccines to metastases, including synthetic, semi-biogenic, and biogenic nanosystems. Moreover, it highlights the physical and pharmacological properties that enhance their anti-metastasis efficiency. This review also addresses the combination of nanovaccines with cancer immunotherapy to target various steps in the metastatic cascade, drawing insights from preclinical and clinical studies. The review concludes with a critical analysis of the challenges and frameworks linked to the clinical translation of cancer nanovaccines.

Epithelial ovarian carcinoma (EOC) is the eighth most common cancer in women and the leading cause of gynaecological cancer death, predominantly due to the absence of effective screening tools, advanced stage at diagnosis, and high rates of recurrence. Circulating tumour cells (CTCs), a rare subset of tumour cells that disseminate from a tumour and migrate into the circulation, play a pivotal role in the metastatic cascade, and therefore hold promise as biomarkers for disease monitoring and prognostication. Exploring CTCs from liquid biopsies is an appealing approach for research and clinical practice, given it is minimally invasive, facilitates serial sampling and enables the capture of the entire spectrum of cancer cells circulating in the blood. The prognostic utility of CTC enumeration has been FDA-approved for clinical use in metastatic breast, prostate, and colorectal cancers. However, the unique biology of EOC, discussed herein, compounds the detection and characterisation complexities already inherent in CTC research, consequently hindering progress towards clinical applications. The aim of this review is to provide an overview of both the biological and clinical challenges encountered in harnessing the power of CTCs in EOC.

Colorectal cancer (CRC) remains a significant global health burden, necessitating a thorough understanding of prognostic and predictive factors to enhance patient outcomes. This systematic review aims to comprehensively evaluate prognostic and predictive determinants in CRC, encompassing both traditional and emerging biomarkers. A systematic search of major electronic databases was conducted to identify relevant studies published from 1995 up to 2024. Eligible articles were critically appraised, and data extraction was performed according to predefined criteria. The prognostic determinants examined included clinicopathological features such as tumor stage, grade, and lymph node involvement, as well as molecular biomarkers including RAS, BRAF, and MSI status. Predictive determinants encompassed biomarkers influencing response to targeted therapies and immunotherapy, such as HER2 and Immunoscore. The review also explores novel prognostic and predictive markers, including tumor microenvironment characteristics and liquid biopsy-based biomarkers. Synthesizing evidence from diverse studies, this review provides insights into the prognostic and predictive landscape of CRC, highlighting the potential clinical implications of identified determinants. Understanding the multifaceted nature of prognostic and predictive factors in CRC is imperative for the advancement of personalized treatment strategies and improvement of patient outcomes.

Neuroendocrine neoplasms (NENs) arise from neuroendocrine cells in a wide variety of organs. One of the most affected disease sites is the gastrointestinal system, which originates the gastro-entero-pancreatic NENs (GEP-NENs), a heterogenous group of malignancies that are rapidly increasing in incidence. These tumors can be functioning, with secretory activity leading to identifiable clinical syndromes, or non-functioning, with no secretory activity but with local symptoms of tumor growth and metastasis. A limitation in biomarkers is a crucial unmet need in non-secretory NEN management, as clinical decision-making is made more difficult by obstacles in tumor classification, prognostic evaluation, assessment of treatment response and surveillance. The objective of this review is to present existing and novel biomarkers for NENs that can function as prognostic factors and monitor disease progression or regression longitudinally, with a special emphasis on innovative research into novel multianalyte biomarkers.

Circulating tumor cells (CTCs) enumeration and molecular profiling hold promise in revolutionizing the management of solid tumors. Their understanding has evolved significantly over the past two decades, encompassing pivotal biological discoveries and clinical studies across various malignancies. While for some tumor types, such as breast, prostate, and colorectal cancer, CTCs are ready to enter clinical practice, for others, additional research is required. CTCs serve as versatile biomarkers, offering insights into tumor biology, metastatic progression, and treatment response. This review summarizes the latest advancements in CTC research and highlights future directions of investigation. Special attention is given to concurrent evaluations of CTCs and other circulating biomarkers, particularly circulating tumor DNA. Multi-analyte assessment holds the potential to unlock the full clinical capabilities of liquid biopsy. In conclusion, CTCs represent a transformative biomarker in precision oncology, offering extraordinary opportunities to translate scientific discoveries into tangible improvements in patient care.

This review delves into the pivotal role of circulating tumor cells (CTCs) in colorectal cancer (CRC) metastasis, focusing on their biological properties, interactions with the immune system, advanced detection techniques, and clinical implications. We explored how metastasis-competent CTCs evade immune surveillance and proliferate, utilizing cutting-edge detection and isolation technologies, such as microfluidic devices and immunological assays, to enhance sensitivity and specificity. The review highlights the significant impact of CTC interactions with immune cells on tumor progression and patient outcomes. It discusses the application of these findings in clinical settings, including non-invasive liquid biopsies for early diagnosis, prognosis, and treatment monitoring. Despite advancements, challenges remain, such as the need for standardized methods to consistently capture and analyze CTCs. Addressing these challenges through further molecular and cellular research on CTCs could lead to improved interventions and outcomes for CRC patients, underscoring the importance of unraveling the complex dynamics of CTCs in cancer progression.

Most studies on CTCs have focused on isolating cells that express EpCAM. In this study, we emphasize the presence of EpCAM-negative and EpCAMlow CTCs, in addition to EpCAMhigh CTCs, in early BC. We evaluated stem cell markers (CD44/CD24 and CD133) and EMT markers (N-cadherin) in each subpopulation. Our findings indicate that all stemness variants were present in both EpCAMhigh and EpCAM-negative CTCs, whereas only one variant of stemness (nonCD44+CD24-/CD133+) was observed among EpCAMlow CTCs. Nearly all EpCAMhigh CTCs were represented by CD133+ stem cells. Notably, the hybrid EMT phenotype was more prevalent among EpCAM-negative CTCs. scRNA-seq of isolated CTCs and primary tumor partially confirmed this pattern. Therefore, further investigation is imperative to elucidate the prognostic significance of EpCAM-negative and EpCAMlow CTCs.

Colorectal cancer (CRC) is a leading cause of cancer mortality globally, underscoring the urgency for a noninvasive and effective biomarker to enhance patient prognosis. Circulating tumor cells (CTCs), a potential marker for real-time tumor monitoring, are limited in clinical utility due to the low sensitivity of existing detection methods. Previously, we introduced a novel nano-based CTCs detection method that relies on the electrical properties of cell surfaces, thus eliminating the need for specific molecular biomarkers. In this study, we used this technique to evaluate the diagnostic and prognostic value of CTCs in stage II-IV CRC.

A total of 194 participants were included, consisting of 136 CRC patients and 58 healthy individuals. The peripheral blood of the participants was collected, and CTC enumeration was performed utilizing the nano-based detection method that we newly developed. The receiver operating characteristic (ROC) curve and multivariate Cox proportional-hazards analysis were used to assess the effectiveness of CTCs for diagnosing CRC and predicting patient prognosis.

The nano-based method demonstrated an ability to differentiate CRC patients from healthy individuals with a sensitivity of 84.6% and a specificity of 94.8%. Furthermore, baseline CTC levels were predictive of progression-free survival (PFS) in CRC patients, with lower levels associated with longer PFS compared to higher levels (4.5 vs 8.0 months at 15 CTCs/mL, p = 0.016; 4.4 vs 8.0 months at 20 CTCs/mL, p = 0.028). We also explored the dynamic changes in the number of CTCs after 1 to 5 cycles of chemotherapy. Patients with increasing CTC levels typically experienced disease progression (PD), while those with decreasing levels often achieved a partial response (PR) or maintained stable disease (SD). These findings suggest that the dynamic fluctuations in CTC counts are closely tied to the clinical course of the disease.

Our study indicates the potential of nano-based CTCs detection in diagnosing and predicting outcomes for patients with stage II-IV CRC.

Circulating tumor cells (CTCs) currently represent a revolutionary tool offering unique insights for the evaluation of cancer progression, metastasis, and response to therapies. Indeed, CTCs, upon detachment from primary tumors, enter the bloodstream and acquire a great potential for their use for personalized cancer management. In this review, we describe the current understanding of and advances in the clinical employment of CTCs. Although considered rare and fleeting, CTCs are now recognized as key players favoring the development of cancer metastasis and disease recurrence, particularly in malignant melanoma, lung, breast, and colorectal cancer patients. To date, the advancements in technology and the development of several successful approaches, also including immunomagnetic enrichment allow for a reliable and reproducible detection and characterization of CTCs. Those innovative methodologies improved the isolation, quantification, and characterization of CTCs from the blood of cancer patients, providing extremely useful evidence and new insights into the nature of the tumor, its epithelial/mesenchymal profile, and its potential resistance to therapy. In fact, in addition to their prognostic and predictive value, CTCs could serve as a valuable instrument for real-time monitoring of treatment response and disease recurrence, facilitating timely interventions and thus improving patient outcomes. However, despite their potential, several challenges hinder the widespread clinical utility of CTCs: (i) CTCs' rarity and heterogeneity pose technical limitations in isolation and characterization, as well as significant hurdles in their clinical implementation; (ii) it is mandatory to standardize CTC detection methods, optimize the sample processing techniques, and integrate them with existing diagnostic modalities; and (iii) the need for the development of new techniques, such as single-cell analysis platforms, to enhance the sensitivity and specificity of CTC detection, thereby facilitating their integration into routine clinical practice. In conclusion, CTCs represent a potential extraordinary tool in cancer diagnostics and therapeutics, offering unprecedented opportunities for personalized medicine and precision oncology. Moreover, their ability to provide real-time insights into tumor biology, treatment response, and disease progression underlines a great potential for their clinical application to improve patients' outcomes and advance our understanding of cancer biology.

Cancer presents a significant challenge to global health, driving worldwide concerted efforts to advance early detection, predict therapeutic response, and identify novel targeted therapies. Liquid biopsies emerge as promising avenues for discerning cancer biomarkers, offering less invasive approaches compared to conventional methods. Utilizing increasingly robust technologies, diverse bodily fluids can unveil genetic variants, epigenetic modifications, transcriptional alterations, and metabolomic signatures associated with cancer, thereby furnishing valuable insights for clinical management. This chapter intends to review the sources of cancer-related biomarkers found in circulation, prevalent techniques utilized for their identification, and the potential implications of different biomarker types on the management of cancer. Certain biomarkers currently used in clinical practice will be addressed, as well as potential biomarkers still in the study phase, and the inherent challenges in their practical implementation.

Early evaluation of tumor heterogeneity related to metastasis and outcomes is a major challenge in the management of advanced breast cancer (BCa) in the clinic. In this study, we introduced the value of baseline circulating tumor cells (CTC) and ctDNA for early differentiation of clinical stages, tumor heterogeneity, and prognosis in clinic.

A total of 292 patients with BCa were enrolled in this study, including 254 Stage IV and 38 Stage III patients, and examined the baseline levels of CTCs, CTC-clusters, and plasma ctDNA before initiating therapies. Outcomes including progression-free survival (PFS) and overall survival were evaluated using proportional hazards regression analysis.

The baseline CTCs, including HER2+ CTCs, in Stage IV patients were approximately 9.5 times higher than those detected in Stage III patients. Baseline CTC counts with a cutoff of 5 were significantly associated with the prognosis. Within each stage, patients with <5 CTCs had significantly longer PFS. Stage III patients with no CTCs exhibited the longest survival compared with patients with ≥1 CTC. CTC-clusters were only found in Stage IV patients, among whom 15 Stage IV patients with ≥5 CTC-clusters had the worst PFS compared with the 239 Stage IV patients with <5 CTC-clusters. Similar outcomes were observed in 28 out of 254 Stage IV patients who had at least one CTC-cluster detected, as these patients had shorter PFS compared with CTC-cluster negative group. The major differences in ctDNA mutations between patients with Stage III and Stage IV BCa were in PIK3CA and ESR1, which were associated with specific organ metastasis and worse outcomes.

Assessing the baseline levels of CTCs, CTC-clusters, and mutational ctDNA profile could reliably aid in differentiation of clinical stage and early prediction of metastasis and outcomes in advanced BCa.

Circulating tumor cells (CTCs) have potential as diagnostic, prognostic, and predictive biomarkers in solid tumors. Despite Food and Drug Administration (FDA) approval of CTC devices in various cancers, the rarity and heterogeneity of CTCs in lung cancer make them technically challenging to isolate and analyze, hindering their clinical integration. Establishing a consensus through comparative analysis of different CTC systems is warranted. This study aimed to evaluate seven different CTC enrichment methods across five technologies using a standardized spike-in protocol: the CellMag™ (EpCAM-dependent enrichment), EasySep™ and RosetteSep™ (blood cell depletion), and the Parsortix® PR1 and the new design Parsortix® Prototype (PP) (size- and deformability-based enrichment). The Parsortix® systems were also evaluated for any differences in recovery rates between cell harvest versus in-cassette staining. Healthy donor blood (5 mL) was spiked with 100 fluorescently labeled EpCAMhigh H1975 cells, processed through each system, and the isolation efficiency was calculated. The CellMag™ had the highest recovery rate (70 ± 14%), followed by Parsortix® PR1 in-cassette staining, while the EasySep™ had the lowest recovery (18 ± 8%). Additional spike-in experiments were performed with EpCAMmoderate A549 and EpCAMlow H1299 cells using the CellMag™ and Parsortix® PR1 in-cassette staining. The recovery rate of CellMag™ significantly reduced to 35 ± 14% with A549 cells and 1 ± 1% with H1299 cells. However, the Parsortix® PR1 in-cassette staining showed cell phenotype-independent and consistent recovery rates among all lung cancer cell lines: H1975 (49 ± 2%), A549 (47 ± 10%), and H1299 (52 ± 10%). Furthermore, we demonstrated that the Parsortix® PR1 in-cassette staining method is capable of isolating heterogeneous single CTCs and cell clusters from patient samples. The Parsortix® PR1 in-cassette staining, capable of isolating different phenotypes of CTCs as either single cells or cell clusters with consistent recovery rates, is considered optimal for CTC enrichment for lung cancer, albeit needing further optimization and validation.

Drug resistance poses a significant challenge in cancer treatment despite the clinical efficacy of cisplatin. Identifying and targeting biomarkers open new ways to improve therapeutic outcomes. In this study, comprehensive bioinformatic analyses were employed, including a comparative analysis of multiple datasets, to evaluate overall survival and mutation hotspots in 27 base excision repair (BER) genes of more than 7,500 tumors across 23 cancer types. By using various parameters influencing patient survival, revealing that the overexpression of 15 distinct BER genes, particularly PARP3, NEIL3, and TDG, consistently correlated with poorer survival across multiple factors such as race, gender, and metastasis. Single nucleotide polymorphism (SNP) analyses within protein-coding regions highlighted the potential deleterious effects of mutations on protein structure and function. The investigation of mutation hotspots in BER proteins identified PARP3 due to its high mutation frequency. Moving from bioinformatics to wet lab experiments, cytotoxic experiments demonstrated that the absence of PARP3 by CRISPR/Cas9-mediated knockdown in MDA-MB-231 breast cancer cells increased drug activity towards cisplatin, carboplatin, and doxorubicin. Pathway analyses indicated the impact of PARP3 absence on the platelet-derived growth factor (PDGF) and G-coupled signal pathways on cisplatin exposure. PDGF, a critical regulator of various cellular functions, was downregulated in the absence of PARP3, suggesting a role in cancer progression. Moreover, the influence of PARP3 knockdown on G protein-coupled receptors (GPCRs) affects their function in the presence of cisplatin. In conclusion, the study demonstrated a synthetic lethal interaction between GPCRs, PDGF signaling pathways, and PARP3 gene silencing. PARP3 emerged as a promising target.

Circulating tumor cells (CTCs) are precursors of cancer in the blood and provide an attractive source for dynamic monitoring of disease progression and tumor heterogeneity. However, the scarcity of CTCs in the bloodstream has limited their use in clinical practice. In this study, we present a workflow for easy detection of CTCs by cytokeratin staining using the FDA-cleared Parsortix device for size-based microfluidic enrichment. To minimize sample handling, the isolated cells are stained inside the separation cassette and harvested for subsequent single cell isolation and whole genome copy-number analysis. We validated the workflow on a panel of four prostate cancer cell lines spiked into healthy donor blood collected in CellRescue or EDTA tubes, resulting in mean recoveries of 42% (16-69%). Furthermore, we evaluated the clinical utility in a cohort of 12 metastatic prostate cancer patients and found CTCs in 67% of patients ranging from 0 to 1172 CTCs in 10 mL blood. Additionally, we isolated single patient-derived CTCs and identified genomic aberrations associated with treatment response and clinical outcome. Thus, this workflow provides a readily scalable strategy for analysis of single CTCs, applicable for use in monitoring studies to identify genomic variations important for guiding clinical therapy decision.

Colorectal cancer (CRC) is one of the most prevalent malignancies affecting the gastrointestinal tract and is ranked third among cancers with the highest incidence and second-highest mortality rate worldwide. CRC exhibits a slow progression providing a wide treatment window. The currently employed CRC screening methods have shown great potential to prevent CRC and reduce CRC-related morbidity and mortality. The diagnosis of CRC is achieved by colonoscopy and tissue biopsy, with studies showing that liquid biopsy is more effective in detecting and diagnosing early CRC patients. Increasing number of studies have shown that the tumor components shed into circulating blood can be detected in liquid form, and can be applied in the clinical management of CRC. Analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), or tumor-associated platelets (TEPs) in the blood can be used for early screening and diagnosis of CRC, aid tumor staging, treatment response monitoring, and prediction of CRC recurrence and metastasis in a minimally invasive manner. This chapter provides an updated review of CTCs, ctDNA, and TEPs as novel biomarkers for CRC, highlighting their strengths and limitations.

Circulating tumor cells (CTCs) have significant potential to become an important tool for monitoring the effects of treatment in solid tumors. The present study reports the occurance of CTCs in cervical cancer (CC) patients during radical chemoradiotherapy (CRT), including brachytherapy (BRT), and during the follow-up period. Patients diagnosed with CC treated with radical CRT were included in the study (n=30). A total of 167 CTC-tests (MetaCell®) were provided at predefined testing time points during the study follow-up (e.g., before CRT, after CRT, every three months of follow-up). In parallel with CTC-testing, SCC-Ag were measured to compare their predictive values during treatment. CTCs were present in 96% (25/26) of patients at the time of diagnosis and in 61% (14/23) after treatment. Patients who relapsed during the 36-month follow-up (n=10) showed an elevation in pre-treatment CTC- numbers, similarly there was a significant increase in pre-treatment SCC-Ag. As next, an increased number of CTCs was observed approximately 12 weeks before relapse was diagnosed by standard imaging modalities (MRI, US, PET-CT) in 3 of 4 patients. In addition to standardized vital cytomorphology of enriched CTCs, quantitative PCR (qPCR) was used to inform the nature of CTCs before treatment. Analysis revealed increased SOX2 and POUSF expression in CTCs in the group of patients with recurrence (P < 0.02). Disease aggressiveness may be related to increased expression of stem cell markers, as found in samples from relapsed patients. CTCs may be an aid to assess tumor burden and disease aggressiveness. An increase in CTCs precedes an increase in SCC-Ag and confirmation of relapse by imaging, as shown in our study.

Appendiceal tumors are uncommon and, at times, discovered incidentally during histological examination. The histopathological classification of the disease is complex and has generated some controversy. The analysis of circulating tumor cells can be used for the early detection of metastatic potential. The aim of the present study was to examine the prognostic value of circulating tumor cells in patients with appendiceal tumors and peritoneal metastases. To our knowledge, this is the first study to examine CTCs in appendiceal tumors. We performed a prospective cohort study of consecutive patients treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy between 2015 and 2019 at a HIPEC referral center. In total, 31 patients were included in the analysis, and circulating tumor cells were detected in 15 patients (48%). CTC positivity was not associated with overall or recurrence-free survival, nor was it correlated with PCI score or histopathological grading. Surprisingly, however, CTCs were found in almost half the patients. The presence or quantities of these cells did not, on their own, predict systemic metastatic potential during the observed time, and they did not appear to significantly correlate with the oncological outcomes recorded.

Circulating tumor cells (CTCs) captured from the bloodstream of patients with solid tumors have the potential to accelerate precision oncology by providing insight into tumor biology, disease progression and response to treatment. However, their potential is hampered by the lack of standardized CTC enrichment platforms across tumor types. EpCAM-based CTC enrichment, the most commonly used platform, is limited by EpCAM downregulation during metastasis and the low EpCAM expression in certain tumor types, including the highly prevalent and lethal NSCLC. In this study we demonstrate that Transferrin Receptor (TfR) is a selective, efficient biomarker for CTC identification and capture in patients with prostate, pancreatic and NSCLC. TfR identifies significantly higher CTC counts than EpCAM, and TfR + -CTC enumeration correlates with disease progression in metastatic prostate and pancreatic cancers, and overall survival and osimetrinib-resistance in non-small cell lung cancer (NSCLC). Profiling of TfR + -CTCs provides a snapshot of the molecular landscape of each respective tumor type and identifies potential mechanisms underlying treatment response to EGFR TKi and immune checkpoint inhibitors in NSCLC.

Transferrin Receptor identifies circulating tumor cells in solid tumors.

Circulating tumor cells (CTCs) play a pivotal role in metastasis, the leading cause of cancer-associated death. Recent improvements of CTC isolation tools, coupled with a steady development of multiomics technologies at single-cell resolution, have enabled an extensive exploration of CTC biology, unlocking insights into their molecular profiles. A detailed molecular portrait requires CTC interrogation across various levels encompassing genomic, epigenetic, transcriptomic, proteomic and metabolic features. Here, we review how state-of-the-art multiomics applied to CTCs are shedding light on how cancer spreads. Further, we highlight the potential implications of CTC profiling for clinical applications aimed at enhancing cancer diagnosis and treatment.

Exploring the complexity of cancer progression through cutting-edge multiomics studies holds the promise of uncovering novel aspects of cancer biology and identifying therapeutic vulnerabilities to suppress metastasis.

The epithelial cell adhesion molecule (EpCAM) is a single transmembrane protein on the cell surface. Given its strong expression on epithelial cells and epithelial cell-derived tumors, EpCAM has been identified as a biomarker for circulating tumor cells (CTCs) and exosomes and a target for cancer therapy. As a cell adhesion molecule, EpCAM has a crystal structure that indicates that it forms a cis-dimer first and then probably a trans-tetramer to mediate intercellular adhesion. Through regulated intramembrane proteolysis (RIP), EpCAM and its proteolytic fragments are also able to regulate multiple signaling pathways, Wnt signaling in particular. Although great progress has been made, increasingly more findings have revealed the context-specific expression and function patterns of EpCAM and their regulation processes, which necessitates further studies to determine the structure, function, and expression of EpCAM under both physiological and pathological conditions, broadening its application in basic and translational cancer research.

Lung cancer is the foremost cause of cancer-related death globally, with non-small cell lung cancer (NSCLC) accounting for 85-90% of cases. Targeted therapy is the most essential therapeutic option for NSCLC, other common treatments include radiation therapy, surgery, chemotherapy, and immunotherapy.

Our study objective was to estimate whether progression-free survival (PFS) is an outcome of NSCLC extracted from 18 randomized control trials (RCTs) with docetaxel as experimental group and antineoplastic agent, kinase inhibitor, and monoclonal antibodies as a control group.

We selected relevant studies published between 2011 and 2022 using Google Scholar, PubMed, Scopus, Science Direct, and Cochrane Library. Advanced NSCLC, chemotherapy, RCT, docetaxel, and second-line treatment were the terms included in the search. A total of 9738 patients were evaluated from the 18 identified studies. We used the meta package of R Studio to perform the meta-analysis. Graphical funnel plots were used to evaluate publication bias visually.

Patients who underwent docetaxel-based therapy had a considerably longer PFS than those who got antineoplastic agents, kinase inhibitors, or monoclonal antibodies-based treatment. Patients in the standard treatment arm had a slightly longer PFS than those in the experimental therapy arm in the overall meta-analysis.

Docetaxel outperformed monoclonal antibodies, antineoplastic agents, and kinase inhibitors in the second-line therapy of advanced NSCLC since PFS was extensively utilized.

Circulating hybrid cells (CHCs) are a newly discovered, tumor-derived cell population found in the peripheral blood of cancer patients and are thought to contribute to tumor metastasis. However, identifying CHCs by immunofluorescence (IF) imaging of patient peripheral blood mononuclear cells (PBMCs) is a time-consuming and subjective process that currently relies on manual annotation by laboratory technicians. Additionally, while IF is relatively easy to apply to tissue sections, its application to PBMC smears presents challenges due to the presence of biological and technical artifacts. To address these challenges, we present a robust image analysis pipeline to automate the detection and analysis of CHCs in IF images. The pipeline incorporates quality control to optimize specimen preparation protocols and remove unwanted artifacts, leverages a β-variational autoencoder (VAE) to learn meaningful latent representations of single-cell images, and employs a support vector machine (SVM) classifier to achieve human-level CHC detection. We created a rigorously labeled IF CHC data set including nine patients and two disease sites with the assistance of 10 annotators to evaluate the pipeline. We examined annotator variation and bias in CHC detection and provided guidelines to optimize the accuracy of CHC annotation. We found that all annotators agreed on CHC identification for only 65% of the cells in the data set and had a tendency to underestimate CHC counts for regions of interest (ROIs) containing relatively large amounts of cells (>50,000) when using the conventional enumeration method. On the other hand, our proposed approach is unbiased to ROI size. The SVM classifier trained on the β-VAE embeddings achieved an F1 score of 0.80, matching the average performance of human annotators. Our pipeline enables researchers to explore the role of CHCs in cancer progression and assess their potential as a clinical biomarker for metastasis. Further, we demonstrate that the pipeline can identify discrete cellular phenotypes among PBMCs, highlighting its utility beyond CHCs.