Isolation and ¹¹¹In–Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model

Abstract

A non-invasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, a little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex-vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice.Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize 111 In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by 51 Cr-release assay. We evaluated physiological distribution of 111 In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and non-irradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology.Results showed that incubation with 0.011 MBq of 111 In-oxine per million murine NK cells in PBS (pH 7.4) for 20-min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while 111 In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24h and 72h p.i., which was accompanied by tumor regression, while in non-irradiated mice, radiolabeled NK cells were retained in the lung tumor lesions up to 72h p.i. without tumor regression. In tumor-bearing mice that were only irradiated but did not receive radiolabeled murine NK cells, a high tumor burden was observed at 72h p.i., which indicates that irradiation in combination with murine NK cell allocation, but not irradiation alone, induced a remarkable anti-tumor effect in the orthotopic A549 lung tumor bearing mouse model.In conclusion, we describe a method to evaluate murine NK cell trafficking and biodistribution, which can be used to determine potential effects of immune-mediated therapeutic agents on NK cell biodistribution.

Manganese-52: applications in cell radiolabelling and liposomal nanomedicine PET imaging using oxine (8-hydroxyquinoline) as an ionophore

“… 5 More recently, it has regained attention for its ability to directly label and track a variety of immune cells. 6 – 11 With the growing availability of PET, focus has shifted towards the development of PET tracers for cell labelling. PET imaging offers higher sensitivity than SPECT imaging as well as improved spatial resolution and quantification in the clinical setting.…”

Characterization of Intrinsically Radiolabeled Poly(lactic-co-glycolic acid) Nanoparticles for ex Vivo Autologous Cell Labeling and in Vivo Tracking

“…Cell labeling with [ 111 In]­In-oxine is fast and with rather high loading efficiency. However, toxicity of oxine and leakage of the radionuclide from the cell are major drawbacks, as they can result in a high background signal during imaging. − Here we have shown that although the NP labeling efficiency is lower, we could achieve similar specific activity per cell and improved radiolabel retention. Radiolabeling of PLGA NPs and other NP platforms have been previously studied for different applications, from in vivo tracking of the NPs to targeting of immune cells for therapeutic purposes. − For example, Sirianni et al used fluorine-18-labeled biotin coupled to avidin-PLGA NPs to measure NP delivery in rat brain with PET .…”

In-vivo imaging of tumor-infiltrating immune cells: implications for cancer immunotherapy

“…They showed that labeling with 0.011 MBq of 111 in-oxine per 10 6 murine NK cells provided optimal labeling efficiency without affecting cell viability or functionality. 94 a total of 1 ×10 6 freshly isolated and labeled cells were then re-infused in a human lung cancer mouse model. Mice with lung cancer showed higher tumor uptake of labeled nK cells than control mice.…”

“…as a control, mice that received no nK cells were irradiated and showed a high tumor burden 72 hours after injection, suggesting that the combination of both radiation and nK cell injection has the best antitumor effect in this mouse model. 94 Passive incorporation in the cell membrane despite the lower translational potential in humans, optical imaging can be performed in pre-clinical models to evaluate NK cell trafficking with no radiation exposure to operator. To this purpose, Tavri et al used near-infrared dye did (1,19-dioctadecyl-3,3,39,39tetramethyl-indodicarbocyanine) to label nK cells targeting epithelial cell adhesion molecule antigen in human prostate cancer xenografts in rats.…”