Pancreatic Circulating Tumor Cells: An Update

Simple Summary

Pancreatic cancer is one of the deadliest cancers, largely because it is difficult to detect early and often returns after treatment. Traditional ways of tracking the disease, like scans and tissue biopsies, can miss important information or be too invasive to use repeatedly. Circulating tumor cells are cancer cells that break away from tumors and enter the bloodstream. Studying these cells through a simple blood test, often called a liquid biopsy, offers a less invasive way to understand how the cancer is growing, spreading, or responding to treatment. This review provides an up-to-date overview of circulating tumor cell research in pancreatic cancer, including how these cells are detected, what they reveal about the disease, and how they could be used to guide treatment decisions in the future. We also highlight current challenges and the promising new technologies that may help bring this approach into everyday cancer care.

Abstract

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by late diagnosis, early metastasis, and poor response to therapy. Liquid biopsy approaches, including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes, offer a minimally invasive method to monitor tumor burden, progression, and treatment response in real time. This review aims to synthesize recent findings on CTCs in PDAC, evaluate detection technologies, and explore their clinical and translational potential. Methods: We conducted a comprehensive literature search using PubMed and Google Scholar, focusing on original studies and reviews published within the past 15 years. Articles were selected based on relevance to CTC biology, detection methods, clinical correlations, and integration with other biomarkers. Attention was paid to studies published since 2018 and landmark earlier works. Results: CTCs are detectable in PDAC patients and are consistently associated with worse survival and higher recurrence rates. However, detection sensitivity varies widely by method. EpCAM-based platforms like CellSearch^® detect CTCs in ~7–48% of cases, while newer size-based and microfluidic approaches report rates above 75%. CTCs exhibit epithelial–mesenchymal and stem-like phenotypes and can form clusters with high metastatic potential. Recent studies demonstrate molecular heterogeneity and show that CTC-derived organoids are feasible for functional studies. Nonetheless, technical variability and the lack of standardization remain major obstacles. Conclusions: CTCs represent a promising biomarker for prognosis and treatment monitoring in PDAC. Further refinement of enrichment techniques, molecular profiling strategies, and prospective clinical validation are needed to integrate CTC assays into routine PDAC management.

1. Introduction

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies, with a five-year survival rate of 13% [1,2,3]. This poor prognosis stems primarily from late diagnosis, rapid metastatic progression, and marked resistance to chemotherapy [1,4]. Only 10–20% of patients present with localized, resectable tumors amenable to curative surgery, and even among those, recurrence rates are high and long-term survival remains limited [1,4,5,6,7]. Because cross-sectional imaging can miss occult micrometastatic disease, preoperative blood-based biomarkers, including CTCs, have been investigated for risk stratification in patients with presumed resectable PDAC [7,8]. These clinical realities highlight the urgent need for better tools to enable earlier detection, stratify prognosis, and monitor disease dynamics in real time. Liquid biopsy has emerged as a promising, minimally invasive approach to address these needs [5,9,10]. Unlike tissue biopsy (e.g., endoscopic ultrasound-guided sampling), which is invasive, cannot be frequently repeated, and may be confounded by tumor heterogeneity and dense stromal tissue [5,11], a liquid biopsy involves analyzing tumor-derived biomarkers in bodily fluids (usually blood). These biomarkers include circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), extracellular vesicles (exosomes/oncosomes), and others. CTCs provide intact cells for phenotyping and functional studies, whereas ctDNA offers sensitive mutation detection; these complementary strengths motivate multi-analyte strategies for monitoring and early relapse detection [9,10]. Blood draws are low-risk and can be done longitudinally, enabling real-time monitoring of tumor dynamics [5]. To date, PDAC has relied on serum CA19-9 as a tumor marker, but CA19-9 is elevated in only ~80% of cases and has limited sensitivity and specificity [12,13,14]. Thus, more robust biomarkers are needed to complement imaging and existing tests.

CTCs are an especially intriguing component of liquid biopsies. These rare cells, shed from primary or metastatic tumor sites into the bloodstream, offer a “window” into the tumor’s biology in real time [15,16]. In fact, CTCs were first observed as far back as 1869 by an Australian physician, Thomas Ashworth [5,16]. However, only in the past two decades have technological advances enabled reliable detection and characterization of these extremely scarce cells [17,18,19]. Research on CTCs has several key objectives: (a) to estimate the risk of metastatic relapse or progression (prognostic value), (b) to monitor treatment response in real time, (c) to identify the therapeutic targets and mechanisms of drug resistance, and (d) to elucidate the mechanisms of metastasis [9,16,20].

That said, studying CTCs in PDAC poses unique challenges such as CTC rarity and detection sensitivity: First, PDAC tumors tend to release very few CTCs compared to other epithelial malignancies [5,16]. Second, PDAC CTC counts are among the lowest of any solid cancer when using standard assays [5,16]. Third, CTCs are a biologically diverse population. Some exhibit epithelial traits, others have mesenchymal or stem-like features, and they can travel as single cells or as clusters [16,21,22]. CTCs undergoing epithelial–mesenchymal transition (EMT) may downregulate epithelial markers like EpCAM, making them harder to detect using traditional capture methods [16,20,23]. This heterogeneity underscores the need for multiplexed or marker-diagnostic approaches to avoid missing entire CTC subpopulations [20]. Fourth, there is currently no single accepted “gold-standard” CTC assay for PDAC [5,9,16]. Techniques vary widely (immunomagnetic, microfluidic, size-based filtration, etc.), each with unique biases and performance characteristics. As a result, different studies report very different CTC detection rates, and direct comparisons between studies are difficult [5]. Fifth, an EpCAM-based assay might miss mesenchymal CTCs, while a size-based method might capture more cells but also include blood cell artifacts [5]. These methodological inconsistencies complicate the interpretation of CTC data [5].

Despite these challenges, accumulating evidence suggests that CTCs hold significant clinical significance in PDAC. Detectable CTCs in PDAC patients are generally associated with more advanced disease and worse prognosis [5,7,8,24,25,26]. As we will discuss, recent studies have correlated the presence of CTCs (especially those with mesenchymal phenotypes or in clusters) with shorter survival and a higher likelihood of recurrence after surgery [5,24]. CTCs also open a path to molecular profiling of PDAC without the need for tissue biopsy, for example, analyzing KRAS mutations or gene expression in CTCs to guide targeted therapies [27,28]. Furthermore, serial CTC enumeration in PDAC has been investigated as a real-time marker of treatment response. Decreases during systemic therapy have been associated with more favorable clinical trajectories, whereas persistent or rising CTC levels may suggest treatment resistance and/or ongoing residual disease [29,30,31,32,33].

The purpose of this review is to provide an up-to-date overview of CTC research in PDAC, with an emphasis on recent advances, ongoing challenges, and future prospects. We first outline the materials and methods used to gather and compare relevant studies. We then present an integrated discussion of CTC biology and clinical applications in PDAC, covering the role of CTCs in PDAC’s metastatic cascade and tumor microenvironment, the evolving technologies for CTC enrichment and detection (from the FDA-approved CellSearch^® system to next-generation microfluidic and imaging platforms), molecular and proteomic characterization of PDAC CTCs, the interplay between CTCs and other liquid biopsy components like oncosomes and ctDNA, and how PDAC CTC findings compare with those in other cancers. Finally, we conclude with perspectives on the path forward, including the need for standardization, the integration of multi-omics data, and the translation of CTC assays into clinical practice.

2. Materials and Methods

Literature Search Strategy: We conducted a comprehensive literature search to identify publications on circulating tumor cells in pancreatic cancer, focusing on articles published in the past ~15 years as well as seminal earlier works. The databases searched included PubMed and Google Scholar (with no language restrictions), using keywords such as “circulating tumor cells,” “CTCs,” “pancreatic cancer,” “PDAC,” “liquid biopsy,” and “circulating tumor DNA.” We sought out both original research studies and review articles that addressed CTC detection technologies, biological characterization of CTCs and clinical correlations in PDAC. Reference lists of relevant papers were also looked at to ensure the inclusion of important studies. Given the rapid advances in this field, we paid special attention to studies from 2018 onward and any highly cited older papers that are frequently referenced in CTC research.

Inclusion of Key Studies: We prioritized high-impact or landmark studies that have shaped current understanding of CTCs in PDAC. For example, the study by Shishido et al. (2024) was included for its innovative analysis of both cellular and acellular tumor-derived components (CTCs and oncosomes) in portal vs. peripheral blood of PDAC patients undergoing surgery [1]. This recent work provided unique insights into how surgical tumor manipulation might acutely influence circulating analytes [1]. We also included the foundational review by Alix-Panabières et al. (2012), which outlined early CTC detection technologies and highlighted the heterogeneity and EMT-associated detection challenges that remain relevant today [34]. Another reference, Trevino et al. (2006), was incorporated as a seminal early study demonstrating that targeting Src kinase activity could inhibit PDAC progression and metastasis in vivo, illustrating the biological pathways underlying metastasis (hence the context in which CTCs operate) [35]. We reviewed Poh & Ernst (2023) for a contemporary perspective on PDAC’s molecular drivers of invasiveness (e.g., c-Src signaling), to connect how an aggressive tumor microenvironment may facilitate CTC dissemination [2]. Additionally, we included Ferrara et al. (2021) for insight into the PDAC stromal microenvironment, given the dense extracellular matrix’s potential impact on CTC release and detection [11].

Data Extraction and Synthesis: We extracted key data points from the selected studies to enable qualitative and (where possible) quantitative comparisons. These data included CTC detection rates in PDAC (percentage of patients in whom CTCs were detected, often stratified by disease stage and method used), assay details (technology platform, enrichment method, markers used), and reported associations between CTCs and clinical outcomes (diagnostic accuracy, prognostic significance such as hazard ratios for survival, etc.). When assembling Table 1, we compared the performance of different CTC detection technologies in PDAC side by side. To do this fairly, we grouped studies by similar methodologies, for example, aggregating results from EpCAM-based immunomagnetic assays like CellSearch^®, and separately summarizing results from filtration or microfluidic approaches, acknowledging that sensitivity and specificity can differ markedly by method [5].

No formal meta-analysis was performed in this review. Instead, we adopt a narrative synthesis approach. We discuss consistent patterns; for instance, nearly all studies agree that presence of CTCs portends worse prognosis in PDAC [24]. We also discuss discrepant findings (e.g., varying CTC detection rates across studies, which we attribute to methodological differences). Throughout the Results and Discussion Sections, we cite specific studies to support each point, and we note the reference source (e.g., clinical study, in vitro experiment, review article) to give context. Literature sources include peer-reviewed journal articles and authoritative reviews; we did not use unpublished data.

Methodological Considerations: It should be noted that the field of CTC research is evolving rapidly. Different studies define “CTC” differently (some require cytokeratin+/CD45 staining for identification, others include vimentin+ mesenchymal CTCs, etc.), and newer techniques are continually emerging. In synthesizing comparisons, we have been careful to account for these differences. For instance, when comparing prognostic impact of CTCs across studies, we consider whether the study looked at any CTC presence vs. absence or at specific CTC counts/phenotypes, and how soon outcomes were measured. Similarly, for detection technologies, we explicitly mention the type of approach and its limitations to contextualize performance differences.

This review was conducted in accordance with the guidelines for narrative reviews provided by the journal Onco (MDPI). All sources are cited in Vancouver style. No human subjects or animals were involved; thus, IRB approval was not required.

3. Results

3.1. Detection Rates and CTC Enrichment Methods in PDAC

CTC detection rates in PDAC vary widely across studies, largely due to differences in enrichment/detection methods and the inherent paucity of CTCs in this disease. Reported detection frequencies range from as low as ~7–11% of patients up to as high as ~90% in some series [5,29]. Notably, an MDPI review of 17 PDAC studies found CTCs in anywhere from 11% to 92% of cases, reflecting heterogeneous methodologies. Simpler techniques like density gradient separation tend to have low yields (CTCs detected in ~24–40% of PDAC patients) [36,37], whereas more advanced approaches have achieved higher sensitivities. For instance, filtration devices (ISET (Boulder, CO, USA), ScreenCell (Paris, France), MetaCell (Cambridge, MA, USA)) report detection in ~66–96% of patients [38,39], and some microfluidic platforms, which capture CTCs in ~75–80% of cases [8,40]. These discrepancies underscore the need for technique standardization, as different studies may not be directly comparable [29]. Indeed, both enrichment strategy and disease stage influence yield, as localized early tumors often release CTCs intermittently if at all, whereas metastatic disease sheds more cells.

One commonly used technology is the CellSearch^® system, an FDA-approved EpCAM-based immunomagnetic enrichment platform considered a gold standard in the CTC field. CellSearch^® has been extensively employed in CTC studies for epithelial cancers, including PDAC (most of the reviewed PDAC studies relied on CellSearch^® for initial capture). However, in pancreatic cancer CellSearch^® has shown limited sensitivity. Multiple studies report PDAC CTC detection rates of only ~7–48% using CellSearch^® (across various stages) despite repeated sampling [26,32,38,41,42,43,44,45]. This is partly because CellSearch^® targets epithelial markers (EpCAM and cytokeratins), which many PDAC CTCs do not robustly express. PDAC tumors often undergo epithelial–mesenchymal transition (EMT), losing EpCAM and other epithelial antigens during dissemination. As a result, EpCAM-negative CTCs, which may be more mesenchymal and aggressive, can evade CellSearch^® detection. Yeo et al. note that EpCAM-based methods can fail to capture a substantial subset of CTCs with low EpCAM, potentially the most invasive subpopulation [5,26,32,38,42,43,44]. To address this, negative selection protocols (removing CD45+ blood cells and leaving any tumor cells) have been applied in PDAC, achieving higher detection rates (up to ~80% in one report) at the cost of possibly discarding some CTCs during handling. Emerging label-independent methods also show promise: for example, high-definition single-cell scanning systems (Epic Sciences, RareCyte) can identify CTCs on slides without EpCAM enrichment. While these imaging-based platforms are FDA-cleared for other cancers, they have not yet been fully tested in PDAC populations. Each approach has trade-offs in sensitivity and specificity, smaller or more deformable CTCs may escape size-based capture, whereas purely marker-dependent methods miss cells that have downregulated the target epitope. Thus far, no universal CTC isolation technique exists for PDAC, and the field lacks a gold-standard protocol [5,26,32,38,42,43,44]. A recent benchmarking study by Macaraniag et al. compared an inertial microfluidic (iMF) device to the immunomagnetic EasySep™ platform for isolating CTCs and CECs in pancreatic cancer. The iMF system achieved approximately 8-fold higher recovery and 5-fold greater enrichment than EasySep™, especially at low cell concentrations, and preserved phenotypic heterogeneity by avoiding reliance on surface markers [46]. The heterogeneity of CTC detection methods and results continues to hinder direct comparison between studies. Ongoing engineering innovations and multimodal approaches (combining physical and immunological capture) aim to improve yield and consistency [5]. Ultimately, standardization and technical advances will be critical for integrating CTC assays into routine PDAC care [5]. To better illustrate the performance characteristics and limitations of CTC detection methods in PDAC, a comparative overview is provided below in Table 1. The following table summarizes key enrichment strategies, typical detection rates reported in PDAC and known limitations in sensitivity and specificity across platforms.

Table 1. Comparison of circulating tumor cell (CTC) detection methods in pancreatic cancer, including enrichment strategy, reported detection rates in pancreatic ductal adenocarcinoma (PDAC), and key limitations.

Method & Principle	Detection Rate in PDAC	Sensitivity/Specificity & Limitations
Density Gradient Centrifugation (e.g., Ficoll separation by density)	~24–40% of patients with detectable CTCs [36,37]	Moderately sensitive (enriches all nucleated cells, including CTCs). Limitations: Low purity: many leukocytes remain; may miss CTCs with overlapping density; labor-intensive manual process [36,37,38].
Filtration (Size-Based) (ISET, ScreenCell micropores ~8 µm)	High: ~66–96% detection in PDAC [38,47]	High sensitivity for larger tumor cells/clusters. Limitations: Can miss small or deformable CTCs [43]; captured cells may include large leukocytes (lower specificity); requires post-filtration staining to confirm identity [43].
Microfluidic Chips (physical or affinity-based microdevices, e.g., CTC-Chip, NanoVelcro, CTC-iChip)	High: ~75–80% in many studies [8,40]	High sensitivity (combines size, flow dynamics, and/or antibody capture); can isolate viable CTCs. Limitations: Many rely on EpCAM or other markers, may miss EMT CTCs if solely marker-based [48]; device optimization needed for consistent results; complex and costly instrumentation [48].
Immunomagnetic Positive Selection (CellSearch® EpCAM-based capture)	Low–Moderate: ~7–48% detection across PDAC stages [26,32,38,41,42,43,44,45].	Clinically validated in other cancers; highly specific for EpCAM+ CTCs; reproducible. Limitations: Low sensitivity in PDAC, misses mesenchymal CTCs [5,26,32,38,42,43,44].; cannot capture CTC clusters or CTCs lacking epithelial markers; requires fluorescent staining (EpCAM, CK, CD45) so viability is lost [48].
Negative Selection (Leukocyte Depletion) (e.g., CD45+ cell removal by magnets)	Moderate–High: ~80% of patients with CTCs in pilot studies [49]	Broad sensitivity can enrich CTCs regardless of surface markers by removing background cells. Limitations: Some CTC loss can occur during depletion; enriched fraction still requires identification of CTCs by cytology or markers; not 100% specific (e.g., rare CD45− normal cells can remain) [49].
High-Definition Single-Cell Assay (HDSCA)	Potentially high [1]	Very sensitive, theoretically detects all CTCs (no bias against EMT phenotypes or clusters). Limitations: Intensive image analysis required; risk of false positives without robust cell characterization; typically uses fixed cells (limited functional downstream assays); not yet standard in PDAC [1].
Combined Multi-Analyte Approaches (e.g., CTC + exosome assays)	Very high in pilot studies: the approach by Buscail et al. achieved 100% sensitivity (with 80% specificity) for PDAC by combining CTC counts with GPC1-exosome detection [42]	Maximizes sensitivity by capturing different tumor-derived signals (cells and vesicles). Limitations: Early-stage research; requires parallel assays and expertise in multiple biomarker types; specificity needs validation (to avoid false positives from benign sources) [5,42].

Abbreviations: CTC = Circulating Tumor Cell; PDAC = Pancreatic Ductal Adenocarcinoma; EpCAM = Epithelial Cell Adhesion Molecule; CK = Cytokeratin; CD = Cluster of Differentiation; ISET = Isolation by Size of Epithelial Tumor Cells; HDSCA = High-Definition Single-Cell Assay; GPC1 = Glypican-1. Detection methods, rates, and limitations summarized from multiple studies [5,26,27,29,36,37,38,40,42,45,46,48,49,50].

Table 1. Comparison of circulating tumor cell (CTC) detection methods in pancreatic cancer, including enrichment strategy, reported detection rates in pancreatic ductal adenocarcinoma (PDAC), and key limitations.

Method & Principle	Detection Rate in PDAC	Sensitivity/Specificity & Limitations
Density Gradient Centrifugation (e.g., Ficoll separation by density)	~24–40% of patients with detectable CTCs [36,37]	Moderately sensitive (enriches all nucleated cells, including CTCs). Limitations: Low purity: many leukocytes remain; may miss CTCs with overlapping density; labor-intensive manual process [36,37,38].
Filtration (Size-Based) (ISET, ScreenCell micropores ~8 µm)	High: ~66–96% detection in PDAC [38,47]	High sensitivity for larger tumor cells/clusters. Limitations: Can miss small or deformable CTCs [43]; captured cells may include large leukocytes (lower specificity); requires post-filtration staining to confirm identity [43].
Microfluidic Chips (physical or affinity-based microdevices, e.g., CTC-Chip, NanoVelcro, CTC-iChip)	High: ~75–80% in many studies [8,40]	High sensitivity (combines size, flow dynamics, and/or antibody capture); can isolate viable CTCs. Limitations: Many rely on EpCAM or other markers, may miss EMT CTCs if solely marker-based [48]; device optimization needed for consistent results; complex and costly instrumentation [48].
Immunomagnetic Positive Selection (CellSearch® EpCAM-based capture)	Low–Moderate: ~7–48% detection across PDAC stages [26,32,38,41,42,43,44,45].	Clinically validated in other cancers; highly specific for EpCAM+ CTCs; reproducible. Limitations: Low sensitivity in PDAC, misses mesenchymal CTCs [5,26,32,38,42,43,44].; cannot capture CTC clusters or CTCs lacking epithelial markers; requires fluorescent staining (EpCAM, CK, CD45) so viability is lost [48].
Negative Selection (Leukocyte Depletion) (e.g., CD45+ cell removal by magnets)	Moderate–High: ~80% of patients with CTCs in pilot studies [49]	Broad sensitivity can enrich CTCs regardless of surface markers by removing background cells. Limitations: Some CTC loss can occur during depletion; enriched fraction still requires identification of CTCs by cytology or markers; not 100% specific (e.g., rare CD45− normal cells can remain) [49].
High-Definition Single-Cell Assay (HDSCA)	Potentially high [1]	Very sensitive, theoretically detects all CTCs (no bias against EMT phenotypes or clusters). Limitations: Intensive image analysis required; risk of false positives without robust cell characterization; typically uses fixed cells (limited functional downstream assays); not yet standard in PDAC [1].
Combined Multi-Analyte Approaches (e.g., CTC + exosome assays)	Very high in pilot studies: the approach by Buscail et al. achieved 100% sensitivity (with 80% specificity) for PDAC by combining CTC counts with GPC1-exosome detection [42]	Maximizes sensitivity by capturing different tumor-derived signals (cells and vesicles). Limitations: Early-stage research; requires parallel assays and expertise in multiple biomarker types; specificity needs validation (to avoid false positives from benign sources) [5,42].

Abbreviations: CTC = Circulating Tumor Cell; PDAC = Pancreatic Ductal Adenocarcinoma; EpCAM = Epithelial Cell Adhesion Molecule; CK = Cytokeratin; CD = Cluster of Differentiation; ISET = Isolation by Size of Epithelial Tumor Cells; HDSCA = High-Definition Single-Cell Assay; GPC1 = Glypican-1. Detection methods, rates, and limitations summarized from multiple studies [5,26,27,29,36,37,38,40,42,45,46,48,49,50].

3.2. Molecular Characterization of CTCs

Beyond enumeration, considerable efforts have focused on molecular profiling of PDAC CTCs to understand their biology and clinical significance. Genetically, CTCs are expected to carry the driver mutations of the primary tumor; indeed, KRAS mutations (present in >90% of PDAC tumors) can often be detected in CTC-derived DNA. Studies comparing CTC and tumor genotypes show both overlap and heterogeneity. For example, Kulemann et al. reported that only ~60% of PDAC CTCs perfectly matched the primary tumor’s mutation profile, with ~40% of patient CTC isolates showing discordance in key mutations like KRAS [27,29,47]. In contrast, Ankeny et al. found 100% concordance of KRAS status between CTCs and the primary tumor in a small series of 5 patients [29,40]. These findings suggest that while CTCs generally recapitulate the tumor’s mutations, there can be genomic heterogeneity, possibly reflecting clonal subsets or additional mutations acquired during metastasis. Detecting KRAS (or other PDAC mutations such as TP53, SMAD4, CDKN2A) in CTCs confirms their malignant origin and can allow for the monitoring of tumor-specific alterations noninvasively [29]. Ongoing improvements in single-cell sequencing have enabled whole-genome or transcriptome analysis of individual CTCs, revealing diverse subclones [30,51]. For instance, genomic analysis of PDAC CTCs has in some cases identified additional mutations or copy number changes not evident in the primary tumor, underscoring the dynamic nature of metastasis (e.g., acquisition of resistance mutations) [5,20,39]. Overall, molecular assays (DNA, RNA, protein) on PDAC CTCs have begun to illuminate which cells are truly metastatic-competent and how they evolve relative to the primary tumor [5,29].

In terms of phenotype, PDAC CTCs display remarkable heterogeneity. A prominent observation is that PDAC CTCs can undergo EMT (epithelial–mesenchymal transition), yielding subpopulations with varying expression of epithelial and mesenchymal markers. Zhao et al. classified CTCs from PDAC patients into three phenotypic categories: purely epithelial CTCs (E-CTCs, positive for EpCAM/cytokeratins only), purely mesenchymal CTCs (M-CTCs, expressing vimentin, twist, etc.), and hybrid epithelial/mesenchymal CTCs (biophenotypic E/M-CTCs) [50]. Using the CanPatrol microfluidic system, they detected CTCs in 78.5% of PDAC patients (107 out of 136 patients), a notably high rate attributable to capturing both epithelial and mesenchymal cells [50]. The total CTC counts ranged widely (0–26 per sample, median 6) [50]. CTC presence and quantity correlated with more aggressive disease: patients with ≥6 CTCs had significantly shorter overall and progression-free survival compared to those with <6 CTCs (median OS/PFS was dramatically worse in the high-CTC group) [50]. Moreover, the presence of mesenchymal CTCs was strongly associated with advanced tumor stage and distant metastasis (patients with M-CTCs were far more likely to have late-stage or metastatic PDAC; p < 0.01) [50]. Patients whose CTCs included a mesenchymal subset tended to have worse prognostic features. This aligns with the notion that EMT confers invasiveness: CTCs that have transitioned toward a mesenchymal phenotype may be the ones capable of seeding new metastatic lesions. Zhao et al. concluded that categorizing CTCs by EMT markers helps identify the most aggressive subpopulations and could inform clinical risk stratification [50]. Consistent with this, other groups have observed that EpCAM-low, vimentin-positive CTCs (i.e., mesenchymal CTCs) are prevalent in PDAC and often correlate with poorer outcomes. PDAC CTCs thus span an epithelial–mesenchymal spectrum, and capturing the full range of phenotypes (not just EpCAM-positive cells) is important for accurate analysis [1,25,29,30,50,52].

Another notable feature of PDAC CTCs is that they can form CTC clusters (also called circulating tumor microemboli). These are multicellular aggregates of tumor cells (sometimes with admixed platelets or leukocytes) that enter the circulation as a group. CTC clusters have been documented in many PDAC patients, particularly those with advanced disease. For example, a 2019 study using ScreenCell filters found CTC clusters (aggregates of ≥3 cells) in 65% of metastatic PDAC patients before starting chemotherapy, and in ~79% of patients after 3 months of treatment [53]. (No such clusters were found in healthy donor controls [53]) Experimental evidence from other cancers suggests that clusters have a higher metastatic potential than single CTCs, cell aggregates are more likely to survive shear stress and immune attack in the bloodstream and successfully colonize distant organs [53,54]. Indeed, clustering of CTCs appears to facilitate metastasis (e.g., by providing survival advantages and cooperative interactions) [53]. In PDAC, the clinical significance of CTC clusters is under investigation. Some studies indicate that patients with CTC clusters tend to have an unfavorable prognosis, e.g., shorter overall survival [55]. What is clear is that PDAC CTC clusters contain viable tumor cells, often displaying epithelial markers, and can also include immune cells [53]. Using immunohistochemistry and confocal microscopy, cluster components in PDAC have been shown to be predominantly CK+/EpCAM⁺ tumor cells, but interestingly some clusters also carry CK+CD45+ hybrid cells, a fusion between macrophages and cancer cells called tumacrophages [55]. This phenomenon of tumor cell–macrophage clusters has shown that in their presence, the advance of PDAC disease correlates significantly [53,56]. Overall, CTC clusters in PDAC are common and represent an important frontier in research, as they likely mark a particularly metastatic subpopulation [53].

Molecular profiling of PDAC CTCs has revealed activation of pathways related to stemness, EMT, and therapeutic resistance. Gene expression analyses on isolated PDAC CTCs demonstrate upregulation of mesenchymal and stem cell markers compared to normal blood cells or even primary tumor tissue. For instance, circulating tumor cells often express EMT transcription factors (such as ZEB1, ZEB2), mesenchymal cytoskeletal proteins (vimentin), and stemness markers like CD44 [53]. In PDAC patients, after chemotherapy, CTCs show elevated expression of stemness/pluripotency markers and developmental pathway genes (e.g., Notch1 and Hedgehog pathway components) [5,57]. High SMO (Smoothened, a Hedgehog pathway protein) expression in CTCs correlates with poor overall survival [5]. While one study linked CD133⁺ CTCs to progressive disease, other analyses found no prognostic value for CD133-positive CTCs [5]. CTCs are molecularly heterogeneous (with subpopulations like EMT-high or stemness-high), and these phenotypes correlate with disease behavior, for example, mesenchymal CTCs are associated with advanced stage/metastasis and stem-like CTCs with therapy resistance [5,50].

Another area of research is the ex vivo culture and expansion of CTCs. Early efforts to culture PDAC CTCs have only yielded short-term organoids: co-culture with immune cells sustained CTC organoids for ~7 days, and a fibroblast co-culture approach was also explored [5]. Research published in 2023–2024 confirms that long-term expandable organoids can be generated from PDAC patient CTCs, a breakthrough that was previously elusive. Tang et al. achieved multi-passage organoids from PDAC CTCs using a tailored 3D co-culture system [58]. These CTC-derived models have been maintained for weeks to months, providing sufficient material for drug screening and molecular analyses that correlate with patient outcomes [58]. The methodologies enabling this progress include advanced CTC isolation techniques (VAR2-based and microfluidic capture), biomimetic culture conditions (supportive stromal factors, 3D matrices, hypoxia), and optimized media formulations [58]. While fully stable 2D CTC lines remain exceedingly rare for PDAC, the advent of CTC-derived organoids offers a practical and arguably superior alternative, preserving tumor heterogeneity in a renewable format [59]. These advances open the door for “liquid biopsy” tumor models that can be used to personalize treatment and investigate metastatic mechanisms in real time [60]. Ongoing challenges include the need for sufficient CTC input and the technical complexity of these culture systems. Nevertheless, the successful establishment of long-term PDAC CTC organoids is a significant step toward integrating CTC-based functional assays into precision oncology for pancreatic cancer [58,59,60].

3.3. Clinical Utility of CTCs in PDAC and Other Malignancies

3.3.1. Prognostic Value in PDAC

A consistent finding across studies is that the presence of CTCs in PDAC is a negative prognostic indicator. Patients with detectable CTCs tend to have more advanced disease and poorer outcomes than those without CTCs. A 2019 review by Martini et al. found that 15 of 17 studies analyzed showed a significant association between CTC positivity and worse overall survival in PDAC. In other words, although methodologies varied, the majority of investigations link CTC detection with shorter survival [29]. Hugenschmidt et al. reported that all patients who had ≥1 CTC detected before surgery subsequently developed distant metastases (usually in the liver) soon after resection, whereas none of the CTC-negative patients developed metastasis as the first recurrence [44]. In that study, CTC positivity was also linked to significantly shorter disease-free survival and post-recurrence survival (hazard ratio ~2.7 for death after recurrence in CTC-positive individuals) [44].

3.3.2. Advanced PDAC

In advanced PDAC (unresectable or metastatic disease), CTC counts have prognostic significance as well. A higher CTC burden usually means more aggressive disease. As noted in Section 3.2, Zhao et al. demonstrated that patients with ≥6 CTCs/7.5 mL had dramatically shorter survival than those with <6 CTCs [50]. Similarly, serial CTC monitoring may predict outcomes. A recent study by Yasui et al. showed that if a patient’s CTC count drops during chemotherapy, the patient tends to have better clinical outcomes, whereas persistently high or rising CTC counts predict disease progression [33]. In that study, patients who achieved a reduction in CTC levels after treatment had improved survival, whereas those whose CTC levels remained elevated or increased had worsening disease, highlighting the potential utility of CTC kinetics as a real-time biomarker of treatment efficacy [33]. This dynamic monitoring aspect, already demonstrated in other cancers, is now emerging in PDAC as well [33]. Overall, the evidence strongly supports that CTCs in PDAC are prognostic, since their mere presence (versus absence) is associated with worse survival, and their quantity or trajectory over time may further refine risk stratification [33]. It is worth noting that CTC positivity also correlates with other adverse clinicopathologic features (such as larger tumor size, lymph node involvement, and metastases) [50], so CTCs reflect tumor burden and aggressive biology. Importantly, CTC status can add information beyond conventional staging. For example, two patients might both have stage III PDAC, but if one has detectable CTCs and the other does not, their outcomes might differ significantly, with the CTC-positive patient at higher risk of rapid progression [44]. Thus, CTCs could help identify high-risk patients who might benefit from more intensive therapy or closer surveillance.

3.3.3. Early Detection and Screening

CTCs have been explored as a potential tool for earlier detection of PDAC, particularly in high-risk populations such as individuals with familial pancreatic cancer or new-onset diabetes [5,29]. Martini et al. and Yeo et al. highlight the possibility of using CTC assays in these groups as part of early screening efforts [5,29]. However, detecting CTCs in early-stage PDAC remains challenging [5,29]. The dense desmoplastic stroma and hypovascular nature of PDAC tumors restrict tumor cell entry into circulation, resulting in very low CTC yields, especially in Stage I–II disease [2,30]. As a result, many localized PDAC patients have undetectable CTCs in peripheral blood [5,29]. Martini et al. note that although CTCs have been identified in patients with precursor lesions or localized tumors, they are not currently a reliable tool for early diagnosis, though future advances in technology may change this [61,62]. To enhance sensitivity, multi-analyte approaches, such as combining CTCs with ctDNA and plasma protein markers, have shown promise [29]. CTCs are also being investigated for postoperative monitoring [5]. Recent prospective data indicate that patients who are CTC-negative after surgery tend to experience longer disease-free survival, whereas those who remain CTC-positive are more likely to experience early recurrence [31].

3.3.4. Comparative Context with Other Malignancies

The concept of using CTCs as a clinical tool originated in other cancers. In diseases like breast, prostate, and colorectal cancer, CTC analysis has been studied for over a decade and has yielded clinically validated insights. For example, in metastatic breast cancer, the CellSearch^® test for CTCs is FDA-cleared and has well-established prognostic value [63]. Similarly, in metastatic prostate cancer and metastatic colorectal cancer, CellSearch^®-based CTC enumeration is prognostic and received FDA approval in those indications [64]. Moreover, molecular characterization of CTCs in other cancers has demonstrates predictive value for guiding therapy [65].

3.3.5. Current Status and Trials in PDAC

It is important to note that no CTC-based test is currently part of routine clinical practice for PDAC [5]. Unlike in breast or colon cancer, where an oncologist might check CellSearch counts [29], in pancreatic cancer CTC assays are still largely research tools [5,29,30]. The predictive value of CTCs in PDAC (i.e., using CTC information to guide therapy choices) remains to be proven [5,29,66]. The fact that sequential CTC changes correlate with treatment response [33] is an encouraging sign that CTCs could be used to monitor therapy in real time. For example, if a patient’s CTC count remains high after a few cycles of chemotherapy, it might indicate resistance and the need to switch regimens, a strategy analogous to what is being tested in metastatic breast cancer management [63]. Clinical trials in PDAC are ongoing to evaluate such uses [30]. Additionally, trials are exploring CTCs as endpoints or biomarkers in the context of new treatments (e.g., does a drug decrease CTC counts, and is that associated with better outcomes?) [5,30]. CTCs are also being studied alongside circulating tumor DNA and exosomes in multimodal liquid biopsy approaches to see if combined biomarker panels can detect recurrence earlier than standard imaging [30].

In summary, CTCs hold considerable clinical promise in PDAC, but their use is still emerging. They clearly carry prognostic information, as numerous studies have shown that PDAC patients with CTCs do worse than those without. There are tantalizing hints of predictive utility (e.g., early changes in CTC levels might predict chemotherapy efficacy), paralleling what is seen in other cancers, but this needs validation in large trials. And while CTCs could theoretically aid early detection or post-surgical surveillance, sensitivity issues currently limit these applications. Nonetheless, CTC research in PDAC is rapidly advancing, fueled by improvements in detection technology and a deeper biological understanding of these cells.

4. Discussion

Research into pancreatic cancer CTCs over the past decade has substantially advanced our understanding of their biology and potential uses but also highlighted significant challenges that must be overcome before CTCs can become a routine clinical tool in PDAC. One major challenge is the technical and methodological heterogeneity discussed in Section 3.1; different studies employ different CTC isolation platforms, each with biases and limitations, leading to widely varying detection rates [5,26,29,32,38,42,43,44,67]. This lack of a standardized, highly sensitive method means that results across studies are not directly comparable, and this impedes clinical translation. Ongoing efforts to develop next-generation enrichment techniques, for example, high-throughput microfluidic devices that reduce reliance on EpCAM (e.g., label-free or multi-marker capture), or novel antibody cocktails that target both epithelial and mesenchymal markers, are critical [16,17,18,19,46,50,66,67]. Further refinement of microfluidic isolation systems, such as minimizing cytocentrifugation-related cell loss as reported by Macaraniag et al., may improve both recovery and downstream analysis feasibility [46]. This highlights that the key barrier is not only sensitivity, but also standardization and analytical validation across platforms, without which results remain difficult to compare and hard to implement clinically [16,19,67].

Another key issue is the biology of CTC release in PDAC [5,29]. Pancreatic tumors are notorious for their dense desmoplastic stroma and poor vascularization [10,11,68,69,70]. This means many tumor cells may not enter the circulation until late in the disease course [5,11,29,68,69,70]. Additionally, both CTCs and ctDNA tend to be present at lower levels in PDAC than in other cancers of similar stage [24,29,30,40,47]. There is also evidence that CTCs may be rapidly lost from peripheral blood through immune interactions and first-pass sequestration/filtration in the hepatic–portal circulation, consistent with higher CTC yields reported in portal venous sampling compared with peripheral blood in some studies [21,71,72,73]. These factors contribute to the low sensitivity of CTC detection in early PDAC and underscore why a negative CTC result cannot definitively rule out the presence of cancer [5,28,29]. It also suggests that combining CTCs with other biomarkers could improve overall detection [5,16,24,29]. For instance, (ctDNA) can capture shedding from tumor cells, which might complement CTC analysis that detects intact viable cells [5,30]. Incorporating exosomes or other secreted vesicles as additional biomarkers may further enhance sensitivity, given that exosomes are abundantly released even by small tumors [5,30,42,74,75]. In essence, the future of liquid biopsy in PDAC will likely involve a multimodal approach, where CTCs are one component among several (ctDNA, exosomal RNA/proteins, etc.), to provide a more comprehensive picture [5,9,16,29,30,34,42,76]. Each analyte has strengths: CTCs allow for cellular analyses and functional testing, ctDNA provides easy-to-detect mutation signals, and exosomes/platelet RNA can reflect tumor activity [5,9,10,29,30]. Together, they could overcome each other’s individual limitations [9,10,30,34,76].

From a clinical standpoint, the most immediately actionable use of CTCs in PDAC is as a prognostic biomarker. The preliminary data are compelling that CTC-positive patients do worse, and thus CTC status could be used to stratify patients in clinical trials or even in practice (e.g., to identify which “early-stage” patients have high risk of micrometastatic disease) [8,41,44,77,78,79]. Some experts envision that a CTC assay could guide therapy intensity, for instance, a patient with resectable PDAC but with CTCs detected pre-surgery might benefit from more aggressive multimodal therapy (neoadjuvant chemo or radiotherapy) because we know upfront that their risk of recurrence is high [44]. In metastatic PDAC, if further research confirms that on-treatment changes in CTC counts correlate with response, then CTC monitoring could become a tool to gauge therapeutic efficacy early [33].

The discussion of CTCs also ties into fundamental questions about PDAC metastasis: Which cells in a pancreatic tumor can metastasize? How early does dissemination occur? By studying CTCs (essentially the “seeds” of metastasis), we gain insight into these questions [80]. Evidence suggests that dissemination in PDAC can occur surprisingly early, even when the primary tumor is small (as indicated by CTCs found in some clinically localized cases) [1,5,23,29,61]. Additionally, the heterogeneity seen in CTC populations (EMT features, clusters, etc.) tells us that metastasis is likely not driven by one homogeneous group of cells, but rather by diverse clones including those that can interact with the microenvironment (e.g., platelets, immune cells in clusters) and resist therapy [53,55]. A particularly intriguing hypothesis is that CTCs might contain a stem-like subpopulation responsible for establishing new metastatic colonies that could potentially lead to the development of new drugs to prevent metastases at the source [77]. In this way, CTC research has implications not just for biomarkers, but for therapeutic discovery [5]. This is a critical point: 90% of cancer deaths are due to metastasis, and in PDAC most patients die from metastatic disease [29,81]. If CTC studies can isolate the culprits of spread, we might find vulnerabilities to exploit.

Looking ahead, there are several future directions for PDAC CTC research: first, integration with other liquid biopsy elements to create robust combined biomarkers for early detection and monitoring; second, technological innovation, e.g., using machine learning to better distinguish CTCs from background cells in imaging assays, or developing point-of-care microdevices that can capture and analyze CTCs from a simple blood draw in real time; third, the development of CTC-derived models. Even though long-term cultures have been elusive [5], advances such as microfluidic tumor-on-a-chip systems or in vivo expansion in immunocompromised mice (CDX models: circulating tumor cell-derived xenografts) might eventually yield stable lines [5,58,82,83,84,85,86,87]. In other cancers such as small-cell lung cancer and breast cancer, CTC-derived xenografts have been created and used to test drugs; a similar approach for PDAC could allow for drug screening of individual patients [5]. Finally, as CTC detection methods improve, screening high-risk groups for PDAC using liquid biopsy might become feasible, perhaps a combined assay (CTCs + ctDNA + CA19-9) applied to populations like those with genetic predisposition or chronic pancreatitis. Though we are not there yet, the concept is being actively investigated [5,88].

As we have reported, there is currently evidence that the presence of CTCs is associated with worse prognosis and a higher risk of rapid progression for PDAC in both surgical [44] and non-surgical patients [25]. The presence of CTC also seems to predict shorter survival in surgical patients as there is a subgroup of CTC-positive patients who did not benefit from the surgical resection of the tumor [44].

Most of the current studies have used the CellSearch^® system for CTC isolation to provide prognostic value and help in directing treatment. We have mentioned in this paper the various limitations of CellSearch^®, even though several studies used and validated this system to provide guidance for prognosis and treatment and different types of cancer such as breast, colorectal, renal, prostate, etc. [26]. It is evident that there is a lack of studies on the role of CTCs in the prognosis of PDAC. To our knowledge, there is only one study that used the CellSearch^® system for the prognosis of PDAC [44] and there is sparse evidence from few other studies mentioned here about the prognostic role of CTCs in PDAC [33,50]. The results of the clinical studies are blunted since cohort composition, stage-mix, sampling site, blood volume and the definition of CTC positivity are not uniform, explaining the discrepancy between the results. In recent years, newer and more effective methodologies have been developed that combine surface markers with cell size separation. Although this is a very challenging task as it is pertinent to many different types of cancers with varied cell biology, we expect that a standardize approach for CTC isolation will be achieved in a timely manner as this field is growing exponentially. Conducting more studies with a standardized methodology will lead to precise results regarding the role of different phenotypes of CTCs in the prognosis and treatment of PDAC but also in premalignant conditions of the pancreas. The evolution of the biochemical parameters of CTCs during various stages of the will be another field of research to elucidate. Future investigations should address this research scope to enable us to obtain relevant and valid information.

5. Conclusions

In conclusion, circulating tumor cells in PDAC represent both a significant opportunity and a formidable challenge. They offer a window into the metastatic process and a means to obtain tumor material without an invasive biopsy. The studies to date establish that CTCs are detectable in many PDAC patients, even early on, and that they carry meaningful prognostic information. There is optimism that, with further research, CTCs could be used to monitor treatment response in real time and perhaps to guide personalized therapies [5,29,89]. Before this can happen, we must improve detection techniques and validate clinical utility in prospective trials. The field is moving quickly: newer technologies are improving sensitivity, and our understanding of CTC biology (EMT, clusters, stemness, etc.) is growing deeper [5]. Encouragingly, what was once a purely experimental observation (tumor cells in blood) is now inching closer to clinical application in PDAC, as it already has in some other cancers [29,64,65,90,91]. We expect that in the future, CTC analysis will be an integral part of personalized medicine for pancreatic cancer, aiding in earlier diagnosis, real-time monitoring, and the discovery of new therapeutic targets to finally improve patient outcomes.