Cancer Letters

Cancer Letters

Volume 403, 10 September 2017, Pages 216-223
Cancer Letters

Original Article
Detection of circulating tumor cells from cryopreserved human sarcoma peripheral blood mononuclear cells

https://doi.org/10.1016/j.canlet.2017.05.032Get rights and content

Highlights

  • Cell-surface vimentin (CSV) is a specific marker to capture CTCs from patient cryopreserved PBMCs across sarcoma tumor types.

  • The novel technology is validated to specifically detect and isolate sarcoma CTCs from cryopreserved PBMCs.

  • This technique allows for the wide use of CTC-based diagnosis and treatment in clinical settings for sarcoma patients.

Abstract

Circulating tumor cells (CTCs) enter the vasculature or lymphatic system after shedding from the primary tumor. CTCs may serve as “seed” cells for tumor metastasis. The utility of CTCs in clinical applications for sarcoma is not fully investigated, partly owing to the necessity for fresh blood samples and the lack of a CTC-specific antibody. To overcome these drawbacks, we developed a technique for sarcoma CTCs capture and detection using cryopreserved peripheral blood mononuclear cells (PBMCs) and our proprietary cell-surface vimentin (CSV) antibody 84-1, which is specific to tumor cells. This technique was validated by sarcoma cell spiking assay, matched CTCs comparison between fresh and cryopreserved PBMCs, and independent tumor markers in multiple types of sarcoma patient blood samples. The reproducibility was maximized when cryopreserved PBMCs were prepared from fresh blood samples within 2 h of the blood draw. In summary, as far as we are aware, ours is the first report to capture and detect CTCs from cryopreserved PBMCs. Further validation in other types of tumor may help boost the feasibility and utility of CTC-based diagnosis in a centralized laboratory.

Introduction

Sarcoma is a rare group of mesenchymal origin tumors, accounting for nearly 20% of pediatric malignancies and less than 2% of adult neoplasms [1], [2]. Despite the low incidence of sarcoma, it represents a much larger proportion in adolescents and young adults with high mortality rate due to late diagnosis and relapse. A potential new approach for the early detection of relapse is to capture the circulating tumor cells (CTCs) from peripheral blood of sarcoma patients who are under remission. CTCs are “seed” cells for tumor metastasis that are shed into the circulatory or lymphatic system from the primary tumor [3], [4]. These cells have attracted attention due to their potential role in early diagnosis and monitoring of therapeutic response to anti-cancer drugs [5], [6], [7]. At present, the CellSearch system is the only technique approved by the US Food and Drug Administration for the detection and enumeration of CTCs in metastatic breast, colorectal, and prostate cancers in the clinical setting [8], [9], [10], [11]. CellSearch captures CTCs by utilizing the epithelial cell adhesion molecule (EpCAM) which, as its name suggests, is overexpressed only in epithelial cancer types [12], [13]. However, this marker is not effective in capturing CTCs originating from mesenchymal tumors such as sarcoma, and might even miss some of CTCs undergoing epithelial–mesenchymal transition (EMT) [12], [14]. Thus, a novel technique for accurately detecting CTCs from sarcoma patients' peripheral blood is quite necessary.

Previously, we have reported that cell-surface vimentin (CSV) is a marker unique to different types of tumor cells [15], [16], [17]. By utilizing CSV as a specific target, we captured and enumerated mesenchymal-derived CTCs and EMT-like CTCs from fresh blood samples of patients bearing different types of cancer with high sensitivity and specificity [14], [18]. However, to the best of our knowledge, the current CTCs capture techniques require fresh blood samples [19]. A reliable and reproducible cytometric technique for the enumeration of CTCs from cryopreserved samples is still lacking. Fresh samples have to be processed within 72 h as collection to maintain the reproducibility [20]. Transportation from multiple laboratories is not only expensive but also may affect the reproducibility of CTCs measurement. All the above barriers limit the application of current CTCs isolation techniques for large multiple-center trials. To boost CTCs assay utility, cryopreserved sample-based CTCs capture should be investigated.

In the current study, we investigated an assay for capturing CTCs from cryopreserved peripheral blood mononuclear cells (PBMCs) from patients with various types of sarcoma using the tumor specific CSV antibody 84-1. The new isolation step can be highly time limiting, which prevents large numbers of samples being processed on the same day. Such a technology will boost the feasibility and utility of CTC-based diagnosis and therapeutic treatment monitoring in large multiple-center trials.

Section snippets

Patient eligibility and recruitment

Patients with metastatic cancer disease were consented in the Department of Laboratory Medicine and Sarcoma Center at The University of Texas MD Anderson Cancer Center. Blood was drawn either before or at least 7 days after intravenous therapy. Blood samples from healthy donors were obtained from Gulf Coast Blood Center in Houston, Texas. The healthy donors had no known disease or infection at the time of blood draw and no history of malignant disease. The study was approved by our

The modified technique enables capture of CTCs from cryopreserved PBMCs

We previously established a method in which enriched CSV-positive CTCs were stained with 84-1 antibody after fixation for immunofluorescence imaging [14]. However, this method was not a feasible approach for cryopreserved PBMC samples. CSV-positive CTCs, captured from cryopreserved samples, stained positive for 84-1 antibody both on the cell surface (CSV) and in the cytoplasm (intracellular vimentin; Fig. 2A). Our previous published results showed that CSV was specific to cancer cells and did

Discussion

In this study, we addressed a technological challenge faced by the entire CTCs research field: how to isolate CTCs from cryopreserved samples? As demonstrated by our findings, we firstly revised the cryopreserved PBMCs preparation for CTCs capture and then modified the established CTCs detection protocol, which was designed for fresh blood samples [15]. In short, we stained the captured CTCs with 84-1 antibody prior to spinning and fixation for immunofluorescent analysis. This is the first

Funding

This work was supported by grants from the National Institutes of Health to Dr. Shulin Li [NIH R01CA120895] and MD Anderson Institutional Research Grant to Dr. Qing H. Meng.

Acknowledgements

Not applicable.

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