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

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.

Keywords: Cell surface vimentin; Circulating tumor cells; Cryopreservation; Peripheral blood mononuclear cells; Sarcoma.

Figure 1. Isolation of human peripheral blood mononuclear cells (PBMCs) and selection of cell-surface vimentin (CSV)-positive circulating tumor cells (CTCs)

(A) Vacutainers used to collect the blood. (B) Buffy coat containing mononuclear cells, neutrophils, smaller numbers of contaminating red blood cells, plasma and platelets. (C) Separation of blood cell types in the SepMate^™ tubes. (D) Schematic representation of 84-1+/CD45- CTCs selection, enumeration and analysis.

Figure 2. Modified technique for CSV marker staining in captured CTCs from osteosarcoma patient and normal PBMCs that have been cryopreserved and thawed

(A–B) Immunofluorescent staining for 84-1 and CD45 antibody in both captured CTCs and normal immune cells after fixation. (C–D) Immunofluorescent staining for 84-1 and CD45 in both captured CTCs and normal immune cells before fixation (non-permeabilized cells). CSV (84-1, green), CD45 (red) and nuclear stain (blue). Scale indicates 10 μm.

Figure 3. Spiking assay results

(A) Micrographs showing CFSE-labeled LM7 cells in the fresh and cryopreserved peripheral blood mononuclear cells from healthy donors. Graphs and tables show the recovery rates for each concentration of spiked cells in the fresh samples (B) and cryopreserved samples (D). The mean results are from at least three independent experiments (error bars indicate standard deviation). Correlations of CTCs counts between the numbers of recovered cells and spiked cells in the fresh samples (C) and cryopreserved samples (E). CSV (84-1, red), tracker dye CFSE (green) and nuclear stain (blue). Scale indicates 10 μm. SD, standard deviation; CV, coefficient of variation.

Figure 4. Comparison of CTCs counts between matched samples

(A–C) Duplicate samples of osteosarcoma PBMCs (N=6) that had been cryopreserved for 2 to 3 years were measured. (D–F) Matched fresh and cryopreserved samples from multiple types of metastatic sarcoma were compared (N=11). (A, D) Immunofluorescent staining for cell surface vimentin and CD45. (B, E) Correlations between CTCs counts for matched samples. (C, F) Bland-Altman plots of the average differences between the matched samples. CSV (84-1, green), CD45 (red), nuclear stain (blue). GIST, Gastrointestinal stromal tumor. Scale indicates 10 μm.

Figure 5. Comparison of CTCs counts within different time lapse before cryopreserving PBMCs

(A) Micrographs showing cell surface vimentin staining in the fresh and cryopreserved samples with different time lapse before cryopreserving PBMCs: less than 2 h, 24h and 48h. Graph shows the results of CTCs counts among fresh sarcoma samples (B) and cryopreserved ones (C) with different time lapse before isolating PBMCs: less than 2h, 24h and 48h. CSV (84-1, green), nuclear stain (blue). GIST, Gastrointestinal stromal tumor. Scale indicates 10 μm. The group of time-lapse less than 2 hours was used as the reference.* P<0.05

Figure 6. Independent tumor markers staining against captured CTCs using modified technique

Captured CSV-positive CTCs validated by the presence of specific mesenchymal markers across 6 samples: α-SMA (red) for osteosarcoma, MDM2 (red) and α-SMA (red) for myxoid liposarcoma, and CD117 (red) for gastrointestinal stromal tumor (GIST). CSV (84-1, green), nuclear stain (blue). GIST, Gastrointestinal stromal tumor. Scale indicates 10 μm.