Isolation Of Viable Circulating Tumor Cells Research Articles

Abstract P2-01-07: Isolation of viable CTCs from leukapharesis product with parsortix system enables subsequent culture

Abstract Background: Solid tumors are constantly releasing circulating tumor cells (CTCs) into the circulatory system. They are genotypically and phenotypically different from the primary tumor. Especially viable CTCs can be of high interest to obtain further therapeutically relevant information. Their extremely low frequency is one of the main limiting factors to use them for further characterization. To overcome this challenge we tested to isolate viable CTCs from diagnostic leukapharesis (DLA) products obtained from breast cancer patients. Material/methods: 16 DLA samples and matched peripheral blood (PB) samples of metastasized breast cancer patients were collected and CTC numbers were determined by CellSearch® analysis. Viable cells were enriched from DLA product with Parsortix system. Genomic DNA of single cultured CTCs was isolated and amplified by whole genome amplification. Array-based comparative genomic hybridization of single cells was performed to analyze for genomic aberrations. Resulting profiles were compared to genomic aberration profiles of CTCs isolated before in vitro culture. Results: CTCs were detected in ten PB and matched DLA samples and in three more DLA samples whose corresponding blood samples were CTC-negative. CTC number per ml was increased by an average of 22.5 fold. Parsortix was optimized to enrich tumor cells from DLA product while keeping them best viable. An enrichment rate of 67.7% ± 11.3% and a harvesting rate of 65.3% ± 9.7% were determined with spiked MCF7 cells. We could grow viable CTCs from three out of eight CTC positive DLA samples from metastasized patients as determined by immune fluorescence analysis. The growing cells harbor genomic anomalies confirming their malignant origin. Most aberrations are widely identical to the aberrations detected in uncultured CTCs. Conclusions: DLA provides greater numbers of viable CTCs which can be enriched with Parsortix system in order to enable their in vitro cultivation. This workflow will allow functional studies. Citation Format: Neubauer H, Franken A, Driemel C, Lampignano R, Behrens B, Reinhardt F, Niederacher D, Stoecklein NH, Fischer JC, Fehm T. Isolation of viable CTCs from leukapharesis product with parsortix system enables subsequent culture [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-07.

Lab on a fabric: Mass producible and low-cost fabric filters for the high-throughput viable isolation of circulating tumor cells

Circulating tumor cells (CTCs) play an important role in estimating the presence and the metastatic relapse of tumor. Despite of their importance, isolation of viable CTCs is still struggling, since chemical or mechanical damages are unavoidable when separating less than 1000 of CTCs out of billions of other blood components. Furthermore, the current CTC isolation devices show low productivity, since they are produced after a series of complicated fabrication processes. Here, we present a low-cost and mass-producible fabric filters for the viable CTC isolation and the further molecular assay for profiling cancer-associated markers. The fabric filter, produced by polyester monofilament yarns, can be massively produced at extremely low-cost, by showing productivity of ~22filters/s at ~59filters/USD. By utilizing size-based sorting method, the fabric filter is capable to isolate both epithelial and mesenchymal CTCs, while slots with curved walls are beneficial for preventing the cell rupture by reducing 21.6% of mechanical stress compared to the conventional straight-walled slots. We applied our filter to 11 human blood samples and found that the number of CTCs was closely related to the expression level of Ki-67, which is highly overexpressed in proliferative tumors. The fabric filter might be an appropriate caner-screening tool in developing countries, where people suffer from insufficient healthcare services.

Abstract 3062: Isolation and characterization of circulating tumor cells (CTCs) from peripheral blood specimens of patients with advanced solid tumor malignancies (using ApoStream™ instrumentation)

Abstract Circulating tumor cells (CTCs) isolated from the peripheral blood of cancer patients provide prognostic information and may inform treatment decisions. Current strategies for CTC isolation and characterization commonly employ epithelial marker-specific antibody capture of fixed cells from blood and detection based on cytokeratin (CK) expression. Our laboratory is currently evaluating an antibody-independent CTC enrichment technology, ApoStream™, which isolates live CTCs from blood, enabled by critical dissimilarities in morphology and dielectric properties of CTCs and blood cells rather than surface marker expression. CTCs isolated with the ApoStream™ instrument are amenable to further high resolution phenotypic characterization, enabling unambiguous identification of enriched cells as malignant cells. We previously reported on the utility of ApoStream™ technology for the isolation of CTCs from patients with advanced alveolar soft part sarcoma (ASPS) with unambiguous confirmation of CTCs by break-apart fluorescent in situ hybridization (FISH) for ASPL-TFE3 gene translocation. Here, we show the isolation of viable CTCs from patients with advanced solid tumors from ongoing clinical studies at the NCI. Since during progression, cancer cells undergo epithelial to mesenchymal transitions (EMT) and mesenchymal to epithelial transitions (MET), we sought to characterize ApoStream™-isolated CTCs using a multiplex phenotyping assay for CD45 (hematopoietic marker), EMT markers (CK, EpCAM, β-catenin and Vimentin), and tumor specific markers (MUC1 and CEA). A user-defined processing algorithm and CTC scoring criteria developed using Definiens® software was used for rare cell detection and enumeration. Our current efforts are focused on evaluating the utility of ApoStream™-isolated CTCs for assessing pharmacodynamic effects of anticancer agents on DNA damage response in patients with refractory solid tumors. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Priya Balasubramanian, Lihua Wang, Scott M. Lawrence, Tony Navas, Shivaani Kummar, Melinda Hollingshead, Francis Owusu, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow, Robert J. Kinders. Isolation and characterization of circulating tumor cells (CTCs) from peripheral blood specimens of patients with advanced solid tumor malignancies (using ApoStream™ instrumentation). [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 3062. doi:10.1158/1538-7445.AM2014-3062

Isolation and expansion of circulating tumor cells (CTC) from melanoma patients using a novel cell culture technique.

10614 Background: Identification of rare (>2-5) circulating tumor cells (CTC) in 7.5 ml blood by immunofluorescence assay (IFA) correlates with a poor prognosis in colon, breast, prostate and lung cancer. Changes in CTC count during treatment also predict the eventual patient progression and survival in these cancers. Existing assays do not detect melanoma CTC, however. In addition, isolation of viable CTC remains problematic. To overcome these limitations we attempted to develop novel melanoma CTC assays, using IFA and cell culture approaches. Methods: Blood samples were obtained from patients and controls following informed consent. The buffy coat (white cells + tumor) was isolated by Ficoll/Hypaque centrifugation, and split into 6 replicate cultures in proprietary TrueCells medium. After 21 days in culture, tumor colonies were counted, and stained for melanoma and leukocyte markers. Buffy coat cells from parallel blood samples were stained with a panel of CSPG4-specific mAb (a pan-melanoma marker) on ultraclean glass slides for analysis by immunofluorescence microscopy. Results: Blood samples were obtained from 16 melanoma patients, ages 28-87. Eight patients were men and 8 were women. CSPG4+ events (>2) were detected in 8/16 patients by IFA (range 0-52). In contrast, tumor cell colonies of >50 cells grew in 12 out of 16 patients with Stage 3 or 4 melanoma (range 0-1054), shown in Table. Cells isolated from CTC colonies produced melanin, stained for CSPG4 and other melanoma markers, but not for leukocyte markers. Control cultures grew no tumor colonies. Conclusions: Our pilot study shows that melanoma CTC can be identified by both IFA and cultured from blood in many patients with stage 3 or 4 melanoma. These CTC exhibited cytologic characteristics diagnostic of melanoma. The culture assay may represent a useful means of enumerating, isolating, and expanding viable melanoma CTC for further molecular study. [Table: see text]

Abstract 3187: Ex vivo characterization of CTCs isolated using the FMSA device

Abstract Although technological advances in recent years have enabled isolatation of circulating tumor cells (CTCs), only the VeridexCellSearch System has been approved by the FDA for assessment of patient prognosis in metastatic breast, colon, and prostate cancers. A fundamental challenge with the CellSearch System is that the technique it uses to enrich and detect CTCs, EpCAM-basedimmunomagnetic isolation, fixates captured cells and prevents further molecular characterization by functional assays and primary cell culture. Progress in addressing these issues may help with progress with a variety of clinical needs including further characterization of the biology of individual patient tumors as well as their potential response top various therapeutics. In this study, we use the Flexible Micro Spring Array (FMSA) Device —a novel portable microfluidic device that captures and isolates viable CTCs by two physical parameters: size and flexibility—to capture, culture, and further characterize CTCs.GFP-labeled HCT-116 cells were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked blood was filtered using the FMSA device, and the recovered cells were expanded in cell culture. A series of experiments were carried out to characterize these cells and investigate the effect of chemotherapy on the resulting cultures. Studies are ongoing to evaluate the potential of using this model to culture CTCs from patients and to evaluate the response of these cells to chemotherapies using high-throughput methodologies. The isolation of viable CTCs from human blood using the FMSA device provides a novel means for studying viable CTCs and for testing drug efficacy ex vivo. The analysis and identification of CTC-specific characteristics and their relation to chemotherapy susceptibility suggests a potential paradigm shift toward personalized and targeted cancer therapy based on the specific genetic defects of each patient's malignancy and CTCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3187. doi:1538-7445.AM2012-3187