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Eur J Nucl Med Mol Imaging (2015) 42:278287

LMIR Lab Meeting Journal Club


March 17th, 2015
Dane Bergstrom

Overview: Cell labeling


Scintigraphic cell tracking used in nuc med for 30
years
Uses autologous WBCs for detecting sites of
infection/inflammation

Traditionally, cells labeled with lipophilic


complexes of 111In
No current clinically used PET tracers for cell
labeling
Saha, GB

Advantages of PET imaging


Higher sensitivity ability to image smaller
number of cells (e.g. eosinophils, neutrophils, Tcells)
Superior resolution better image quality
Enhanced quantification

Purpose of this study


To develop and evaluate a 89Zr PET tracer for cell
labeling and imaging and compare to gold
standard ([111In]oxinate3 SPECT)

Methods overview
Synthesis of [89Zr]oxinate4
In vitro measurement of uptake/efflux in three cell lines + human
WBCs
In vivo biodistribution in mice of 89Zr- or 111In-labeled myeloma
cells
In vivo retention of 89Zr in myeloma cell line in C57 black mice by
FACS
Comparative nanoPET/CT ([89Zr]oxinate4) and nanoSPECT/CT
([111In]oxinate3) imaging in mice

[89Zr]oxinate4

89

oxine ligand

Zr oxalate pH-adjusted to neutral aqueous solution

Added to a vessel containing oxine dissolved in chloroform


Vessel shaken x 15 minutes; [89Zr]oxinate4 recovered from
chloroform by phase evaporation
QC showed radiochemical purity >99%:
ITLC-SG (ethyl acetate mobile phase)
[89Zr]oxinate4 Rf = 0.9; [89Zr]oxalate Rf = 0

Biphasic solvent system (PBS & chloroform 1:1)

Cell labeling

1 106 cells in 450 L of culture medium


Add [89Zr] label; incubate for up to 60 minutes at room temperature
Centrifuge, remove supernatant, and count supernatant and pellet
separately in gamma counter

Cellular efflux
Cells labeled with [89Zr]oxinate4 or [111In]oxinate3 and incubated in
culture medium at 37C
Supernatant & pellets counted at various time points up to 24 h
Retention of 89Zr in WBCs
after 24h (n = 2):
85.1%
86.9%

Cell viability
Evaluated [89Zr]oxinate4-labeled murine myeloma cells by
trypan blue dye exclusion 24 hours after labeling
Before labeling: 93% viability
Immediately post-labeling: (P = 0.01)
[89Zr]oxinate4: 76.3 3.2%
Control: 89.7 1.2%

24 h post-labeling: (P 0.05)
[89Zr]oxinate4: 89.7 4.0%
Control: 89.3 3.0%

Ex vivo cell tracking 2 days


Mice inoculated iv with [89Zr]oxinate4-labeled eGFP-5T33 myeloma
cells

Ex vivo cell tracking 7 days


Mice inoculated iv with [89Zr]oxinate4- or [111In]oxinate3-labeled
eGFP-5T33 myeloma cells
Biodistribution of each evaluated by ex vivo tissue sampling 7 days
post-injection

Ex vivo cell tracking FACS


Mice inoculated with [89Zr]oxinate4-labeled eGFP-5T33 cells and
sacrificed at 2 and 7 days
Liver, spleen, femora harvested
Samples sorted into eGFP+ and eGFP- for gamma counting

In vivo cell tracking


Mice injected iv with 107 eGFP-5T33 cells labeled
with 5 MBq [89Zr]oxinate4
PET/CT performed at 1, 2, 5, 7 & 14 days pi

Mice injected iv with 107 eGFP-5T33 cells labeled


with 10 MBq [111In]oxinate3
SPECT/CT performed at 1, 2, 3, 5 & 7 days pi

Control groups:
[89Zr]oxinate4 (no cells)
Lysed [89Zr]oxinate4- or [111In]oxinate3-labeled cells

PET/CT & SPECT/CT

89

Zr-oxine 5T33
14 d

PET/CT & SPECT/CT Controls

Discussion

Characteristics of an ideal tracer

?
?

Suitably long physical t


Appropriate positron energy/abundance
Convenient/economic availability
Simple/efficient labeling
No major cell type selectivity
Minimal effects on cell survival
Minimal efflux from cells

Critiques (1) Uptake and Retention


Methods mention the evaluation of uptake in human WBCs, but only retention
data at one time point is presented; no information in the cell labeling section
Knowing how well human WBCs take up tracer is critically important for clinical
translation

In the Efflux from cells section, authors claim that for all three labeled cell
lines, 89Zr efflux was significantly slower than 111In, but no P-values are
reported
Also no data for 111In-oxine retention in the murine myeloma cell line eGFP-5T33

Authors refer to cellular efflux without discussing mechanism by which


radionuclide is ejected
Better title: Cellular retention
This is the title they used for the corresponding figure

Critiques (2) Viability


Cell viability: assessed the murine myeloma cell line, not human
WBCs?
Claim that survival of 89Zr-labeled 5T33 cells is not significantly
different to that of control cells
Viability is reduced to 76.3% immediately post-labeling with 89Zr
(significantly worse than control), but then viability 24 hours postlabeling is higher, at 89.7%; how is this possible?
Unclear methodology; potentially misleading

Claim that survival of 89Zr-labeled 5T33 cells is comparable to or


better than 111In-labeled cells
No viability data for 111In-labeled cells in this paper

Thoughts & future directions


Seems promising
Analogous procedure to labeling cells with 111In would enable
quick clinical implementation
Slightly longer half-life and superior sensitivity of PET imaging
may be able to provide a superior alternative to 111In-WBC
labeling, especially at later timepoints
Dosimetric studies needed (acknowledged by authors)
Studies in different cell types

Slide 1 of 4

Ketamine use Review


Our use of ketamine (component of the animal
anesthetic cocktail for injection) is governed by
the Controlled Drugs and Substances Act, 1996
Necessitates strict safeguarding, including
maintaining inventory and secure storage
Please adhere to the following steps for proper
ketamine usage

Slide 2 of 4

Ketamine use Review


Transporting to animal facility:
Transport in Rx vial to prevent cracking of
vial and loss of inventory (any inventory
loss needs to be reported to Health
Canada)
Always wipe vial lid with alcohol swab
(with friction) and allow to air dry before
withdrawing dose
Follow AUP for dose and route of
administration; record doses administered
Usually 4 L of cocktail per gram of body
weight for mice

Slide 3 of 4

Ketamine use Review


Returning to the lab inventory sheet and
storage:
Find the inventory sheet corresponding to the
anaesthetic vial that was used to anaesthetize
the animals
Record all the required information on the
inventory sheet
Write the the total dose of anaesthesia cocktail
administered to all mice anaesthetized that
day

Return the anaesthetic vial and inventory


sheet to the metallic lock box in the top
drawer in the northwest corner of the main
lab

Slide 4 of 4

Ketamine use Review


Please follow these steps
to ensure that we are in
compliance with the
pertinent federal
legislation
Thank you