Fluorine-19 Labeling of Stromal Vascular Fraction Cells for Clinical Imaging Applications

Abstract

Stromal vascular fraction (SVF) cells are used clinically for various therapeutic targets. The location and persistence of engrafted SVF cells are important parameters for determining treatment failure versus success. We used the GID SVF-1 platform and a clinical protocol to harvest and label SVF cells with the fluorinated ((19)F) agent CS-1000 as part of a first-in-human phase I trial (clinicaltrials.gov identifier NCT02035085) to track SVF cells with magnetic resonance imaging during treatment of radiation-induced fibrosis in breast cancer patients. Flow cytometry revealed that SVF cells consisted of 25.0% ± 15.8% CD45+, 24.6% ± 12.5% CD34+, and 7.5% ± 3.3% CD31+ cells, with 2.1 ± 0.7 × 10⁵ cells per cubic centimeter of adipose tissue obtained. Fluorescent CS-1000 (CS-ATM DM Green) labeled 87.0% ± 13.5% of CD34+ progenitor cells compared with 47.8% ± 18.5% of hematopoietic CD45+ cells, with an average of 2.8 ± 2.0 × 10¹² ¹⁹F atoms per cell, determined using nuclear magnetic resonance spectroscopy. The vast majority (92.7% ± 5.0%) of CD31+ cells were also labeled, although most coexpressed CD34. Only 16% ± 22.3% of CD45-/CD31-/CD34- (triple-negative) cells were labeled with CS-ATM DM Green. After induction of cell death by either apoptosis or necrosis, >95% of ¹⁹F was released from the cells, indicating that fluorine retention can be used as a surrogate marker for cell survival. Labeled-SVF cells engrafted in a silicone breast phantom could be visualized with a clinical 3-Tesla magnetic resonance imaging scanner at a sensitivity of approximately 2 × 10⁶ cells at a depth of 5 mm. The current protocol can be used to image transplanted SVF cells at clinically relevant cell concentrations in patients.

Significance: Stromal vascular fraction (SVF) cells harvested from adipose tissue offer great promise in regenerative medicine, but methods to track such cell therapies are needed to ensure correct administration and monitor survival. A clinical protocol was developed to harvest and label SVF cells with the fluorinated (¹⁹F) agent CS-1000, allowing cells to be tracked with (19)F magnetic resonance imaging (MRI). Flow cytometry evaluation revealed heterogeneous ¹⁹F uptake in SVF cells, confirming the need for careful characterization. The proposed protocol resulted in sufficient ¹⁹F uptake to allow imaging using a clinical MRI scanner with point-of-care processing.

Keywords: Breast cancer; Cell tracking; Fluorine; Liposuction; Magnetic resonance imaging; Radiation-induced fibrosis; Stromal vascular fraction.

©AlphaMed Press.

Figures

Figure 1.

Development of clinical stromal vascular fraction (SVF) harvest protocol. Flow cytometry histograms show DAPI staining, with DAPI-positive cells gated (A). The cells were incubated in lactated Ringer’s solution (Ai), phosphate-buffered saline (PBS) (Aii), or Hank’s balanced salt solution (HBSS) (Aiii) for 2 hours. SVF cell pellets were transferred from GID SVF-1 to conical tubes after one (Bi) or two (Bii) washes, and before (Biii) and after (Biv) red blood cell removal. Samples of tryptic soy broth (TSB) from process simulation (Ci) and standards ([Cii] left, negative control; middle, positive control; right, negative control) were incubated to determine bacterial contamination. Note the cloudiness of TSB only in the positive control. Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride.

Figure 2.

Development of clinical stromal vascular fraction (SVF) labeling protocol. (A): SVF cells were labeled with 20 mg/ml CS-ATM DM Green for 4 hours in either the PBS or the DMEM and analyzed with flow cytometry after ammonium-chloride-potassium (ACK) treatment. (B): Percentage of 4′,6-diamidino-2-phenylindole dihydrochloride-positive (viable) cells for the same PBS and DMEM groups shown in (A). (C): To ensure that the choice of red blood cell (RBC) removal method did not affect 19F labeling, SVF cells were treated with either ACK lysis buffer or gradient centrifugation to remove RBCs. The SVF cells were then labeled in DMEM with 20 mg/ml CS-ATM DM Green for 4 hours and analyzed with flow cytometry. (D): Labeling was confirmed with fluorescent microscopy (scale bar = 100 μm). Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline.

Figure 3.

Stromal vascular fraction (SVF) cells labeled with CS-ATM DM Green. SVF cells labeled with dual mode CS-ATM DM Green (fluorescein isothiocyanate channel) were stained with anti-CD45 (allophycocyanin channel) and anti-CD34 (phycoerythrin channel) antibodies. Representative flow cytometry images show unstained CS-ATM DM Green-labeled cells (A, D), unlabeled cells stained with anti-CD34 (B) or anti-CD45 (E), and CS-ATM DM Green-labeled cells stained with anti-CD34 (C) or anti-CD45 (F).

Figure 4.

Fate of 19F after induction of cell death. 19F-labeled cells were exposed to staurosporine or freeze/thaw cycles to induce apoptotic and necrotic cell death, respectively, with untreated cells as the control. After 4 days, dead cells and cell debris were harvested by centrifugation, washed, and analyzed by nuclear magnetic resonance (NMR) for 19F content. Shown is the total 19F content for three independent runs, which were pooled to create sufficient signal for NMR detection.

Figure 5.

19F 3-Tesla magnetic resonance imaging (MRI) of labeled stromal vascular fraction (SVF) cells in a silicone breast phantom. (Ai): Diagram outlines the six cell injection variables (0.5, 1.0, and 2.0 × 106 cells injected at either 5- or 10-mm depth) and CS-1000 standards. (Aii): An optical image of the final injected breast implant phantom. Coronal (Aiii) and sagittal (Aiv)1H MR images of the phantom show nonspecific contrast from injection sites. (Bi–Biii): Sequential 19F MR images of the phantom show MRI signal only for 2.0 × 106 cells injected at a 5-mm depth. (C): This was confirmed by overlaying the cell and standard diagram on the slice from (Bii).