Original ArticleDetection of circulating tumor cells from cryopreserved human sarcoma peripheral blood mononuclear cells
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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2020, Coordination Chemistry ReviewsCitation Excerpt :Another angle that bioanalytical scientists have been paid attention to is the combine of multiple enrichment strategies. Rather than focusing on the surface markers of CTCs, isolation of matrix cells based on their surface markers have been successfully applied for pre-enrichment, one well-accepted example is CD45 on leukocytes [90,91]. However, when some impurity shares the same biomarkers with CTCs such as cellular debris, this strategy may seem not sufficient.
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2019, Translational ResearchCitation Excerpt :This CSV-mAb requires live cell staining before permeabilization to distinguish between PBMCs and tumor cells. This revised CSV-based capture method is able to detect CTCs from cryopreserved PBMC samples as well.45 Ongoing work, as part of clinical trials involving neuroblastoma patients is expected to shed further light on the validity and reliability of our approach.
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Isolation of circulating tumor cells in a preclinical model of osteosarcoma: Effect of chemotherapy
2018, Journal of Bone OncologyCitation Excerpt :Using a method based on isolation by size of tumor cells (ISET), Chinen et al. [29] detected CTCs in sarcoma patients. More recently, Li et al. [30] confirmed the detection of CTCs in cryopreserved human peripheral blood mononuclear cells and two studies analysing CTCs by FISH in small cohorts of osteosarcoma patients revealed a potential positive association between CTC count, disease progression and poor prognosis [31,32]. The present report is the first study investigating the regulation of CTC release after chemotherapy.