Size-based Circulating Tumor Cells Enrichment Research Articles

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

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

Abstract 3370: Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer

Abstract Aggressive variants of prostate cancer (AVPC) emerge from prostate adenocarcinoma following enduring therapeutic pressure. They grow independent of the androgen receptor, progress rapidly and can show traits of neuroendocrine (NE) differentiation. As progression to AVPC is not well detected by prostate specific antigen monitoring, new biomarkers are urgently needed. The aim of this study is to evaluate the detection of circulating tumor cells (CTCs) in combination with gene expression analysis of NE genes to identify AVPC patients with neuroendocrine traits. Blood samples were collected from patients suffering from AVPC (n=78, including 25 patients with histological evidence of NEPC) as well as from patients with hormone-sensitive prostate cancer (HSPC) as controls (n=12) at the University Medical Center Hamburg-Eppendorf. CTC counts were determined by CellSearch analysis. In parallel, CTCs were immuno-magnetically enriched using the AdnaTest and bulk gene expression of prostate-specific and NE genes was analyzed by semi-quantitative PCR. From a subset of patients, CTCs were additionally enriched by Parsortix to compare gene expression of CTCs with EpCAM-based or size-based CTC enrichment. Using CellSearch analysis, CTCs were found in 88.9 % of AVPC patients. CTC counts ranged between 0 – 20,000 CTCs/7.5 ml of blood with a median of 32 CTCs/7.5 ml. Similarly, 81.8 % of NEPC patients were positive for CTCs with a median count of 35 CTCs/7.5 ml (range 0-13,000). In contrast, CTCs were found in only 50 % of HSPC patients at lower concentrations (median count of 0.5 CTCs/7.5 ml, range 0-34). CTC counts were significantly elevated in AVPC and NEPC patients compared to HSPC (p = 0.002, p = 0.012), but no significant differences were found between AVPC and NEPC samples.In 93.8 % of AVPC samples, CTCs expressed prostate-specific genes such as KLK3 or PSMA. At least one of the NE transcripts was detected in 37.5 % of AVPC patients. In CTCs from NEPC patients, the positivity for prostate markers was significantly reduced to 66.6 % (p = 0.01) and the positivity for NE markers was increased to 61.1 %. A random Forrest model trained on all analyzed transcripts allowed for a distinction of HSPC and NEPC with an AUC of 79.5 %. Comparison of CTC enrichment strategies in a subset of 13 patients showed similar NE marker detection by size-based enrichment (53.8 % NE-positive) compared to the AdnaTest (61.5 % NE-positive). AVPC and NEPC patients show a high CTC burden that will allow subsequent molecular analyses, and CTC counts allowed a distinction between blood samples from HSPC and NEPC patients. Gene expression analysis revealed a high degree of inter-patient heterogeneity for neuroendocrine-specific transcripts with a reduction of prostate markers in NEPC patients. Future molecular characterization and longitudinal monitoring of CTCs will shed more light on the evolution of AVPC and NEPC. Citation Format: Lina Merkens, Sarah Greimeier, Sabine Riethdorf, Klaus Pantel, Gunhild von Amsberg, Stefan Werner. Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3370.

Abstract 6705: Molecular detection of circulating cancer cells with cancer stem cell or mesenchymal characteristics in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is a clinically challenging disease that requires a multidisciplinary approach. Unfortunately, the scarcity of ESCC genomic data hinders the understanding of ESCC biology, disease progression and rational therapy design. Circulating tumor cells (CTCs), which are shed from the primary and metastatic tumors and then circulate within the peripheral vasculature, reflect the existing tumor heterogeneity. Here, we utilized the non-biased size-based CTC enrichment strategy in combination with the real-time RT-PCR to molecularly characterize the CTCs with cancer stem cell (CSC) or mesenchymal properties in ESCC. Comprehensive data from ESCC cell lines, mouse ESCC xenograft models and clinical ESCC peripheral blood samples emphasize the importance of TWIST1 (Twist Family BHLH Transcription Factor 1) in ESCC progression. Gain-of-function and loss-of-function analyses demonstrate that TWIST1 promotes cell migration, invasion and colony formation in ESCC cells. Moreover, positive TWIST1 expression is responsible for the chemoresistance of ESCC cells to Cisplatin and is correlated with poor overall survival in ESCC patients. In addition, we show that TWIST1 promotes malignant potential, including tumor growth, invasion and chemoresistance via the TWIST1-TGFBI-ZEB1 signaling pathway in ESCC. Meanwhile, the TWIST1-TGFBI-ZEB1 signaling pathway confers immunosuppressive conditions to the tumor microenvironment, which in turn contributes to EMT to promote tumor progression. Collectively, these findings indicate that TWIST1 has a novel role in ESCC carcinogenesis by regulating tumorigenicity and cancer stem cell properties that may be useful as a prognostic monitoring biomarker and a promising therapeutic target in clinical ESCC treatment. Citation Format: Zhen Tan, Josephine Mun-Yee Ko, Valen Zhuoyou Yu, Hoi Yan Ng, Simon Law, Maria Li Lung. Molecular detection of circulating cancer cells with cancer stem cell or mesenchymal characteristics in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6705.

Pre-Analytical and Analytical Variables of Label-Independent Enrichment and Automated Detection of Circulating Tumor Cells in Cancer Patients.

Circulating tumor cells (CTCs) are promising tools for risk prediction and the monitoring of response to therapy in cancer patients. Within the EU/IMI CANCER-ID consortium, we validated CTC enrichment systems for future inclusion into clinical trials. Due to the known heterogeneity of markers expressed on CTCs, we tested the Parsortix® system (ANGLE plc) which enables label-independent CTC enrichment from whole blood based on increased size and deformability of these tumor cells compared to leukocytes. We performed extensive comparisons both with spiked-in blood models (i.e., MDA-MB-468 tumor cell line cells spiked at very low concentration into blood from healthy donors) and validated the protocol on actual clinical samples from breast, lung, and gastrointestinal cancer patients to define optimal conditions for CTC enrichment. Multiple parameters including cassette gap, separation pressure, and cell fixatives were compared in parallel. Also, the compatibility of blood collection tubes with whole genome amplification of isolated tumor cells was demonstrated and we furthermore established a workflow for semi-automated CTC detection using a quantitative cell imager. The established workflow will contribute to supporting the use of size-based CTC enrichment platforms in clinical trials testing the clinical validity and utility of CTCs for personalized medicine.

Nucleus of Circulating Tumor Cell Determines Its Translocation Through Biomimetic Microconstrictions and Its Physical Enrichment by Microfiltration.

The mechanism of cells passing through microconstrictions, such as capillaries and endothelial junctions, influences metastasis of circulating tumor cells (CTCs) in vivo, as well as size-based enrichment of CTCs in vitro. However, very few studies observe such translocation of microconstrictions in real time, and thus the inherent biophysical mechanism is poorly understood. In this study, a multiplexed microfluidic device is fabricated for real-time tracking of cell translocation under physiological pressure and recording deformation of the whole cell and nucleus, respectively. It is found that the deformability and size of the nucleus instead of the whole cell dominate cellular translocation through microconstrictions under a normal physiological pressure range. More specifically, cells with a large and stiff nucleus are prone to be blocked by relatively small constrictions. The same phenomenon is also observed in the size-based enrichment of CTCs from peripheral blood of metastatic cancer patients. These findings are different from a popular viewpoint that the size and deformability of a whole cell mainly determine cell translation through microconstrictions, and thus may elucidate interactions between CTCs and capillaries from a new perspective and guide the rational design of size-based microfilters for rare cell enrichment.

Abstract 1713: Transferrin receptor 1 (TfR) as marker for circulating tumor cells (CTCs) identification in NSCLC

Abstract CTCs are used as a surrogate source of tumor material in solid tumors. Clinical applications of CTCs as liquid biopsy comprise the monitoring of molecular alterations during tumor progression and dynamic evaluation of molecular markers of treatment response. The FDA-cleared method to isolate CTCs in cancer patients (Cell Search) is based on positive selection of EpCAM expressing cells. However, this approach performs poorly in non-small cell lung cancer (NSCLC) as it identifies CTCs in only 7% of the subjects, failing to show any prognostic relevance. Down-regulation/loss of epithelial markers to isolate (EpCAM) and identify (cytokeratin, CK) CTCs could in part explain the low CTC yield obtained in NSCLC with approaches based on epithelial markers expression. To overcome this challenge we used size-based CTC enrichment (ISET filters) from NSCLC patients’ peripheral blood. As a positive identifier of CTCs we used transferrin receptor 1 (TfR) which is a cell membrane-associated protein, that mediates intracellular iron uptake, and which is expressed at low levels in many normal tissues but over-expressed in cancer cells. We first analyzed TfR protein expression by immunofluorescence in a panel of NSCLC cell lines and in healthy donor leukocytes. While all NSCLC cells lines analyzed were positive for TfR expression, none of the leukocyte expressed the receptor. Moreover, TfR expression was detected also in EpCAM negative NSCLC cell lines. To determine the clinical applicability of this novel CTC identifier, we determined TfR expression in CTCs isolated from peripheral blood of 35 metastatic NSCLC patients using the ISET filter technology. The isolated CTCs were stained for TfR, CK, CD45 and DAPI. For each patient, one additional ISET filter was stained with Giemsa for morphologic analysis by a pathologist. By using the classic panel of CTC identifiers markers (CK+/CD45-/DAPI+), CTCs were identified in 4/34 (11%) patients, while by using TfR as positive identifier (TfR+/CD45-/DAPI+) CTCs were identified in 31/35 (88%) subjects. The morphologic review of Giemsa stained filters confirmed the presence of tumor cells in 28/34 (82%) samples [0-217 CTCs/sample]. Interestingly, patients with &amp;gt; 6 TfR+ CTCs had a worse overall survival (OS) than patients with &amp;lt; 6 TfR+ CTCs [p=0.048 Log Rank (Mantel-Cox)]. OS did not significantly differ using the same cutoff with CTCs defined based on CK or Giemsa staining. Overall, our data indicate that TfR is a promising biomarker for the detection of CTCs in NSCLC CTCs, superior to CK or EpCAM. Our data also suggest that TfR may potentially identify CTCs subpopulations with a significant prognostic role in NSCLC. We are currently isolating TfR+ CTCs from early stage and metastatic NSCLC patients for further molecular characterization and determination of clinical significance. Citation Format: Giuseppe Galletti, Galatea Kallergi, Ashish Saxena, Despoina Aggouraki, Christos Stournaras, Vassilis Georgoulias, Timothy E. McGraw, Nasser Altorki, Paraskevi Giannakakou. Transferrin receptor 1 (TfR) as marker for circulating tumor cells (CTCs) identification in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1713. doi:10.1158/1538-7445.AM2017-1713

A multiplexed marker-based algorithm for diagnosis of carcinoma of unknown primary using circulating tumor cells.

Real-time, single-cell multiplex immunophenotyping of circulating tumor cells (CTCs) is hypothesized to inform diagnosis of tissue of origin in patients with carcinoma of unknown primary (CUP). In 20 to 50% of CUP patients, the primary site remains unidentified, presenting a challenge for clinicians in diagnosis and treatment. We developed a post-CellSearch CTC assay using multiplexed Q-dot or DyLight conjugated antibodies with the goal of detecting multiple markers in single cells within a CTC population. We adapted our approach to size-based CTC enrichment protocols for capturing CTCs and subsequent immunofluorescence (IF) using a minimal set of markers to predict the primary sites for common metastatic tumors. The carcinomas are characterized with cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) or prostate-specific antigen (PSA. IF has been optimized in cultured tumor cells with individual antibodies, then with conjugated antibodies to form a multiplex antibody set. With IF, we evaluated antibodies specific to these 5 markers in lung, breast, colorectal, and prostate cancer cell lines and blood from metastatic prostate and breast cancer patients. This advanced technology provides a noninvasive, diagnostic blood test as an adjunct to routine tissue biopsy. Its further implementation requires prospective clinical testing.

Abstract 1600: A multiplexed marker-based algorithm for diagnosis of carcinoma of unknown primary using circulating tumor cells

Abstract Abstract: Real-time, single-cell multiplex immunophenotyping of circulating tumor cells (CTCs) is hypothesized to inform diagnosis of tissue of origin in patients with carcinoma of unknown primary (CUP). In 20 to 50% of CUP patients, the primary site remains unidentified, presenting a challenge for clinicians in diagnosis and treatment. Invasive tissue biopsy is the gold standard for diagnosis but improved methodology is needed. We developed a post-CellSearch CTC assay using multiplexed Q-dot or DyLight conjugated antibodies with the goal of detecting multiple markers in single cells within a CTC population. We adapted our approach to size-based CTC enrichment protocols for capturing CTCs and subsequent immunofluorescence (IF) using a minimal set of markers to predict the primary sites for common metastatic tumors. The carcinomas are diagnosed with cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) or prostate-specific antigen (PSA) as the minimal set of informative markers. IF has been optimized in cultured tumor cells with individual antibodies, then with conjugated antibodies to form a multiplex antibody set. With IF, we evaluated antibodies specific to these 5 markers in lung, breast, colorectal, and prostate cancer cell lines and blood samples from metastatic prostate and breast cancer patients. Our technological advance provides a noninvasive, inexpensive, diagnostic blood test as an adjunct to routine tissue biopsy. Its further implementation requires prospective clinical testing. Citation Format: Elizabeth M. Matthew, Lanlan Zhou, Namrata Vijayvergia, David T. Dicker, Karen S. Gustafson, Harry S. Cooper, Eric A. Ross, Bora Lim, Ramdane Harouaka, Si-Yang Zheng, Nicholas E. Lamparella, Joseph J. Drabick, Cristina I. Truica, Zhaohai Yang, Wafik S. El-Deiry. A multiplexed marker-based algorithm for diagnosis of carcinoma of unknown primary using circulating tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1600. doi:10.1158/1538-7445.AM2015-1600

Abstract 4822: Immunophenotyping circulating tumor cells in patients with unknown primary

Abstract Metastasis is responsible for the majority of cancer-related deaths. A limiting step in metastasis is access to circulation, thus circulating tumor cells (CTCs) should be present in all patients with metastatic tumors, which may have both diagnostic and prognostic value. Accounting for only 3-5%, carcinoma of unknown primary (CUP) presents a challenge for clinicians in diagnosis and therapy. In 20 to 50% of CUP patients, the primary remains undiagnosed even after a full diagnostic work up. Tissue biopsy is the standard for diagnosis but there is need for improved methodology, including a non-invasive blood test that relies on CTC analysis. Our overall objective is to utilize CTCs to provide insight into systemic cancer progression and approaches to therapy, including a CTC blood test as a tool for diagnosis, prognosis and therapy. We hypothesize that real-time, single-cell immunophenotype profiling of CTC populations will inform diagnosis of tissue of origin, reveal prognostic markers such as stem-like properties and predict tumor aggressiveness. We have been developing a post-Veridex CTC analysis method using multiplexed Q-dot conjugated antibodies with the goal of detecting multiple markers in each single cell within a CTC population to aid in CUP diagnosis including tissue of origin. We have adapted this method for use in CTCs captured using the flexible micro spring array (FMSA) size-based CTC enrichment protocol and other approaches for capturing and immunophenotyping live CTCs. Further adaptation of this protocol enables the use of Dye-Light conjugated primary antibodies. For carcinomas we use cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor (TTF-1), estrogen receptor (ER; for female patients) and prostate specific antigen (PSA; for male patients) as a minimal set of markers for the identification of lung, colon, prostate and breast cancer. Successful validation of antibodies to these five markers has enabled multiplexing after DyLight conjugation in positive and negative cell lines. This approach is currently being tested in CTCs from patients and is being expanded to understand the biology of CTCs through an evaluation of EMT and stem cell markers. Successful application of our CTC-CUP protocol to CTCs in patient samples is expected to provide a noninvasive, relatively quick and inexpensive blood test that can be both diagnostic and prognostic, which may be a valuable adjunct to routine biopsy. Circulating tumor cells may provide insight into the mechanism of systemic cancer progression, the characteristics of metastatic founder cells and changes within these cells as cancer progresses, thereby impacting the efficacy and evaluation of cancer therapy. Citation Format: Elizabeth M. Matthew, Lanlan Zhou, Bora Lim, Nicholas E. Lamparella, David T. Dicker, Jean-Nicolas Gallant, Monika Joshi, Sheldon L. Holder, Ramdane Harouaka, Siyang Zheng, Joseph J. Drabick, Cristina I. Truica, Zhaohai Yang, Wafik S. El-Deiry. Immunophenotyping circulating tumor cells in patients with unknown primary. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4822. doi:10.1158/1538-7445.AM2014-4822

Abstract 5104: Isolation, culture and immunophenotyping of live patient-derived circulating tumor cells.

Abstract Metastasis is responsible for the majority of cancer-related deaths. A limiting step in metastasis is access to circulation, thus circulating tumor cells (CTCs) should be present in all patients with metastatic tumors. Tissue biopsy is the standard for diagnosis of carcinoma of unknown primary (CUP) and other metastatic carcinomas but there is a need for improved methodology. Circulating tumor cells not only enable real-time monitoring of cancer progression and response to therapy but may be utilized to provide personalized treatment for patients by evaluating the patients own CTCs in culture with therapeutics prior to systemic administration. Our overall objective is to utilize CTCs to provide insight into systemic cancer progression and approaches to therapy, including a CTC blood test as a tool for diagnosis, prognosis and therapy. We hypothesize that isolation, culture and real-time, single-cell immunophenotype profiling of CTC populations will inform diagnosis of tissue of origin, reveal prognostic markers such as stem-like properties, predict tumor aggressiveness and enable evaluation of potential therapies. We have been developing a post-Veridex CTC analysis method using multiplexed Q-dot conjugated antibodies for detection of multiple markers in each single cell within a CTC population to aid in CUP diagnosis. In parallel, we are developing a flexible micro spring array (FMSA) size-based CTC enrichment protocol and other approaches for capturing, culturing and immunophenotyping live CTCs. We have demonstrated that as few as 20 cancer cells spiked into blood may be captured with the FMSA device and grown into the millions of cells necessary for evaluating potential therapies. Due to the challenges of growing patient-derived tumor cells in culture, minimal progress has been made in this area. Cells captured from a patient with adenocarcinoma of unknown primary that have proliferated in culture on an FMSA device are being be evaluated for CK8,18 expression to identify putative CTCs. Utilizing an alternate approach, we have successfully captured and cultured CK8,18 positive and CD45 negative putative CTCs from the blood of a colorectal cancer patient. In addition to developing our approach for diagnosis of tissue of origin, we will utilize the FMSA device and other approaches for isolating live CTCs to study the biological properties of these cells in the context of metastatic spread. Successful application of our protocol to CTCs in patient samples is expected to provide a noninvasive, relatively quick and inexpensive blood test that can be both diagnostic and prognostic, and provide a platform for personalized therapeutic strategies. Circulating tumor cells may provide insight into the mechanism of systemic cancer progression, the characteristics of metastatic founder cells and changes within these cells as cancer progresses, thereby impacting the efficacy and evaluation of cancer therapy. Citation Format: Elizabeth M. Matthew, Jean-Nicolas Gallant, Lanlan Zhou, Zhaohai Yang, Joseph J. Drabick, Cristina I. Truica, Nicholas E. Lamparella, David T. Dicker, Wafik S. El-Deiry. Isolation, culture and immunophenotyping of live patient-derived circulating tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5104. doi:10.1158/1538-7445.AM2013-5104