Fig 1.
Schematic representations of the ApoStream® device.
(A) ApoStream® prototype instrument. The device has a width of 24 inches, and a height of 17 inches. The scale bar represents 12 inches. (B) 3D CAD model of the flow chamber showing V-shaped injection and collection ports. The chamber measures 6 inches (length), by 2 inches (height), by 2 inches (depth). The scale bar represents 2 inches. (C) Step 1: Sample processing by Ficoll density gradient separation to isolate PBMCs and CTCs. Step2: DEP enrichment starting with sample injection, ion diffusion, DEP separation of CTCs from PBMCs, and CTC collection. The scale bar represents 2 inches. Step 3: downstream analysis using immunofluorescence or other techniques for CTC identification and enumeration.
Fig 2.
Representative images of CTCs from metastatic ASPS patients.
(A-B) Specimens were probed with a two-color (green and red) probe set (for additional details, see Online Methods) and counterstained with DAPI. Co-localization of the probes (visualized as a yellow signal), indicates a normal Xp11.2 locus. Yellow arrowheads denote cells with TFE3 rearrangements (separate green and red signals), which identify CTCs characterized by the separation of the probes. Scale bars indicate 20 μm.
Table 1.
Summary of spiked cancer cell experiments at 2 sites.
Table 2.
TFE3 break-apart FISH analysis of ASPS specimens.
Fig 3.
Enumeration and phenotypic characterization of CTCs from patients with sarcoma.
Blood from advanced sarcoma patients and healthy donors were processed using the ApoStream® device. Healthy donors (n = 9, open circles) were used to demonstrate assay specificity. (A) In 4 of the 15 sarcoma patients, phenotypic heterogeneity was seen. Colored dotted lines correspond to the assay cut-off for each of the phenotype measured. STN, soft tissue neoplasm; ES, embryonal cell sarcoma; LS, liposarcoma; CS, chondrosarcoma; LMS, leiomyosarcoma; SS, synovial sarcoma; STS, soft tissue sarcoma. (B) The advanced sarcoma patients (n = 15, closed circles) had significantly higher numbers of cells with CD45-/VIM+ phenotype (p = 0.0002), with 73% of patient samples above the 21 cells/mL cut-off. (C) Gallery of CTCs from a patient with soft tissue neoplasm (STN; top) and a patient with embryonal cell sarcoma (ES; bottom). Putative CTCs isolated using ApoStream® were identified using antibodies against CK/β-catenin (orange), vimentin (red), and a nuclear stain (DAPI), while excluding leukocytes with marker CD45 (green). Scale bars correspond to 20 μm.
Fig 4.
Validation of TLE1 as a tumor-specific marker of sarcoma.
TLE1 (Transducin-Like Enhancer Protein 1) was identified as tumor-specific marker for sarcomas. (A) Western blot analysis of TLE1 (MW ~83 kDa) in: 1: sarcoma cell lines; 2: melanoma cell line; 3: PBMCs. GAPDH (MW ~37 kDa) served as a loading control. (B) CTCs isolated from the blood of a patient with sarcoma and characterized by TLE1 (yellow), vimentin (green), and DAPI (blue), while excluding CD45+ leukocytes (red) staining (4 channels); representative 80X images are shown. Scale bars indicate 20 μm. (C) Determination of false positive CD45-/Vimentin+/TLE1+ cells in a panel of healthy blood donors.
Table 3.
Phenotypic characterization of putative CTCs with vimentin and TLE1.