Translation of high-field fluorine-19 cell tracking techniques into the clinical realm
Jeff M Gaudet1,2, Corby Fink3,4, Matthew S Fox1, Gregory A Dekaban3,4, and Paula J Foster1,2

1Imaging Research Laboratories, Robarts Research Institute, London, ON, Canada, 2Medical Biophysics, Western University, London, ON, Canada, 3Molecular Medicine, Robarts Research Institute, London, ON, Canada, 4Microbiology and Immunology, Western University, London, ON, Canada

Synopsis

Cellular MRI can be used to improve outcomes of cancer immunotherapy by tracking the fate of these cells after their administration. In this study, we used fluorine-19 MRI to track and quantify migration of antigen-presenting peripheral blood mononuclear cells (PBMC). Mice were imaged at both high-field and clinical field strengths. PBMC migration to the node was quantified and compared under different conditions. This study is the first to report on fluorine-19 imaging of PBMC and demonstrates the potential of cellular MRI to aid in the optimization of cellular therapy.

Background:

Cancer vaccine-based therapies are an area of expanding research. In 2010, the first FDA approved cell therapy was released to target metastatic castration-resistant prostate cancer1. This therapy uses antigen-presenting peripheral blood mononuclear cells (PBMC) in order to prime the immune system to target a tumor. Despite this progress, clinical results have been varied and inconsistent. Patient outcome is strongly dependent on in vivo behavior of these cells after administration. Antigen containing PBMC must migrate to a nearby draining lymph node to exert their therapeutic effect. Fluorine-19 (19F) cellular MRI offers a tool to non-invasively track the fate and quantify migration of these cells. 19F-MRI provides unambiguous detection, with a signal that is linearly dependent on the number of cells/voxel. In this study we: (i) used a pre-clinical high field MRI system to track PBMC with 19F, (ii) implemented 19F imaging of PBMC on a clinical 3T MRI system.

Methods:

Human PBMC were labeled with a commercial perfluorocarbon agent. NMR was performed on a known number of labeled cells to determine the average PBMC 19F uptake. Flow cytometry was used to determine the percentage and cell type taking up the 19F agent. Migration of individual cell types to the node was assessed by magnetically sorting T-cells, B-cells and monocytes from the PBMC mixture. Individual cell types were uniquely tagged with a fluorescent marker. Cells were recombined in physiological proportions, injected into mice, and 48hrs later fluorescent microscopy was performed on ex vivo lymph nodes. For in vivo MRI studies, 3x106 PBMC were administered into the footpad of nude mice and imaged after 48hrs. Mice were imaged on a 9.4T small animal scanner using a custom-built, dual-tuned 19F/1H birdcage volume coil. A balanced steady state free precession (bSSFP) sequence was used at a resolution of 1x1x1mm3 for 19F and 200x200x200μm3 for 1H. The number of PBMC migrating to the draining lymph node was compared between four groups: (1) PBMC matured in culture with granulocyte macrophage colony-stimulating factor (GM-CSF), (2) pre-treatment of the lymph node with the pro-inflammatory agent interleukin-1β (IL-1β), (3) both GM-CSF and IL-β, and (4) non-treated controls. Quantification was performed by measuring the 19F signal in regions of interest and in a reference tube of known 19F concentration.

Translational potential was demonstrated on a 3T system with a dual-tuned surface coil (4.3x4.3cm2) designed and approved for human use. 19F-bSSFP images were acquired of three PBMC cell pellets ranging from 1-10x106 cells at a resolution of 0.5x0.5x1cm3 and a scan time of 15min. In vivo imaging was performed following injection of 15x106 PBMC into the mouse hindlimb muscle using the same parameters.

Results/Discussion:

NMR showed no difference in 19F uptake between GM-CSF matured PBMC (1.8±1.2x1011 19F/cell) and control PBMC (1.8±1.4x1011 19F/cell) Figure 1A]. Flow cytometry revealed that 99.4% of PBMC take up the 19F-agent, and that all cell types are labeled. [Figure 1B-C]. 19F labeling of both B- and T-cells was observed. We were also able to show that all 3 cell types migrate to the lymph node following injection [Figure 2]. With MRI, in vivo detection of PBMC was observed in the draining lymph node of mice 48 hours post footpad injection [Figure 3A]. Ex vivo analysis of these lymph nodes confirmed migration of human labeled PBMC [Figure 3B]. 19F signal within the lymph node was quantified to determine the number of migrating PBMC [Figure 4]. Signal was detectable in 22/30 nodes when GM-CSF and/or IL-1β treatments were used. By comparison, migration was only detected in 1/10 nodes from control animals. This result suggests pre-treatment with either agent is important for promoting consistent migration over the 19F-MRI detection threshold. There was no significant difference in the number of migrating PBMC as determined with 19F-MRI.

At 3T all cell pellets were detectable within the 15min scan session [Figure 5A-C]. In the smallest pellet, as few as 4x1017 19F/spins were detectable, the highest sensitivity reported in the literature with a clinical 3T system thus far2. In vivo detection of 15x106 PBMC was observed following intramuscular injection into a mouse [Figure 5D-E].

Significance:

This study is the first to report on imaging of PBMC. Here we have shown the highest reported sensitivity thus far for fluorine labeled cells using a clinical protocol. In addition, this study demonstrates the potential for 19F-MRI to aid in the pre-clinical optimization of cellular therapy. Based upon these results, we anticipate translation of 19F-MRI into the clinic is feasible and foresee application of the technique in the near future for tracking PBMC in cancer patients.

Acknowledgements

This study was supported by the Smarter Imaging Program from the Ontario Institute for Cancer Research (OICR) and by a Movember Discovery Grant from Prostate Cancer Canada.

JMG is funded by the Sam Ciccolini Graduate Studentship from Prostate Cancer Canada and the London Cancer Research and Technology Transfer (CaRTT) training program. JMG and CF are members of the Western Molecular Imaging collaborative program.

References

1.PROVENGE (sipuleucel-T) [statistical review and evaluation]. Seattle, WA, USA: Dendreon Corp; 2010. US Food and Drug Administration website. http://www.fda.gov/BiologicsBloodVaccines/CellularGeneTherapyProducts/ApprovedProducts/ucm210215.htm

2. Ahrens ET, Helfer BM, O'Hanlon CF, and Schirda C. Clinical cell therapy imaging using a per fluorocarbon tracer and fluorine-19 MRI. Magnetic Resonance in Medicine 2014;72(6):1696-701

Figures

19F labels all cell types comprising PBMC. (A) Average 19F uptake of the bulk PBMC was found to vary between donor. There is no difference in 19F uptake between cells matured with GM-CSF and untreated controls. (B) 99.4% of bulk PBMC are labeled with the 19F agent. (C) All three cell types which comprise PBMC are efficiently labeled with 19F. Optimum uptake across cell types was found when labeling at a concentration of 5x106 cells/mL and with 5mg/mL of 19F agent

All components of PBMC migrate to the lymph node. (A) B cells, T cells, and monocytes were isolated from bulk PBMC mixture. (B) Each cell type was labeled with a different fluorescent tag. The three cell types were recombined in physiological proportions as a PBMC mixture, prior to administration into the mice. 48hrs after injection mice were sacrificed and the lymph node was extracted. (C) Ex vivo fluorescent microscopy reveals migration of all three tagged cell types to the node.

19F MRI allows for in vivo tracking of PBMC (A) 48hrs after administration of 3x106 PBMC, signal is observed in the popliteal lymph nodes (yellow arrows). 19F-signal quantification reveals the number of migrating PBMC. (B) MRI was confirmed by ex vivo flow cytometry analysis of the lymph node. Prior to injection, human PBMC were labeled with CFSE, a cytoplasmic fluorescent agent lost on cell death. Presence of this signal with human CD45 confirms live PBMC migration to the lymph nodes.

Quantification of PBMC migration. The number of migrating PBMC was compared between four conditions: (1) PBMC matured in culture with GM-CSF. (2) IL-1β, a proinflammatory cytokine, administered to prime the lymph node. (3) Both GM-CSF matured cells and node pre-treatment with IL-1β. (4) Control with no treatments. A significant difference was found in the frequency of detection between treated and untreated controls. Strong 19F signal was visible at the site of injection in the footpad.

Feasibility of clinical translation. (A)3 PBMC pellets were imaged at 3T, using our custom, human-approved surface coil. Pellets were produced with clinically relevant numbers ranging from 1-10x106 PBMC. (B)19F-MRI reveals all 3 pellets, (C)overlaid onto the proton image. (D)With the same coil, in vivo proton imaging was acquired after injection of 15x106 PBMC into the hindlimb of a mouse. (E)19F-MRI shows the site of injection, (F) given anatomical context when overlaid on the proton image.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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