Imaging Lung Clearance of Radiolabeled Tumor Cells to
Study Mice with Normal, Activated or Depleted Natural
Killer (NK) Cells
P.V. Kulkarni, M. Bennett, A. Constantinescu, V. Arora, M. Viguet, P. Antich,
R.W. Parkey, D. Mathews, R.P. Mason and O.K. Oz
Departments of Radiology and Pathology, The University of Texas Southwestern Medical Center at
Dallas, Texas 75390
Abstract. Lung clearance of 51CR and 125I iododeoxyuridine (IUDR) labeled cancer cells assess NK cell activity. It is
desirable to develop noninvasive imaging technique to assess NK activity in mice. We labeled target YAC-1 tumor cells
with 125I, 111In, 99mTc, or 67Ga and injected I.V. into three groups of BALB/c mice. Animals were treated with medium
(group I), 300mg/kg cyclophosmamide (CY) to kill NK cell (group II), or anti-LY49C/l) (ab’)2 mAb to augment NK
function (group III). Lungs were removed 15 min or 2 h later for tissue counting. Control and treated mice were imaged
every 5 min with a scintillating camera for 1 h after 15 min of infusion of the 111In labeled cells. Lung clearance
increased after 15 min (lodging: 60-80%) and (2 h retention: 3-7%). Similar results were obtained with all the isotopes
studied. Images distinguished the control and treated mice for lung activity. Cells labeled with 111In, 99mTc or 67Ga are
cleared similar to those labeled with 51Cr or 125I. NK cell destruction of tumor cells may be assessed by noninvasive
imaging method either by SPECT (99mTc, 111In, 67Ga) or by PET (68Ga).
C.B-17 SCID NK cells. Thus, blocking Ly49
inhibitory receptors can enhance resistance to H2identical or syngeneic tumor cells in vitro and in vivo
(1). The short half-life of F(ab')2 mAbs limits their
efficacy in vivo, suggesting that non-depleting whole
mAbs are needed. This is the first definitive evidence
that NK inhibitory receptors are responsible for
diminishing anti-tumor responses, and the data suggest
that the strategy of blocking these receptors
continually
may
increase
the
success
of
immunotherapy.
INTRODUCTION
The ability of neoplastic cells to evade the
immune system remains a formidable barrier limiting
the success of immunotherapy. In mice subsets of NK
cells express inhibitory and/or stimulatory Ly49
receptors for MHC class I determinants. Although NK
cells can kill tumor cells in vitro and in vivo, tumor
cells may evade NK cell tumor surveillance by
expressing class I molecules recognized by inhibitory
Ly49 receptors. Perhaps blocking such interactions
could enhance anti-tumor effects of NK cells. Because
35-40% H2b B6 NK cells express Ly49C and/or Ly49I
receptors recognized by 5E6 mAbs, this large subset
should not be able to kill syngeneic tumor cells well.
We used 5E6 F(ab')2 mAbs to block Ly49C/I - H2b
class I (Kb) interactions to determine if NK cell antitumor effects were enhanced. This reagent (i) inhibited
the ability of C1498 leukemia cells to inhibit IFNg
secretion by NK cells, (ii) enhanced the lysis of C1498
and EL4 cells in vitro by 5E6+ NK cells, (iii) inhibited
colony formation by C1498 and EL-4 in vitro, and (iv)
enhanced survival of B6 mice challenged with C1498
leukemia. 4D11 F(ab')2 mAbs that recognize Ly49G2,
an inhibiting receptor for H2-Dd, blunted the ability of
H2d P815 tumor cells to inhibit INFg secretion by H2d
METHODS
Cytotoxicity Assay
1.5-2 x 106 YAC-1 lymphoma cells (prototypic
target cells for murine NK cells) were grown in
complete RPMI and were diluted to low concentration
to force them into log phase growth the night before
the assay. The YAC-1 cells were incubated for 1.5
hours at 37° C in a total volume of 0.6 ml with 150250 µCi sodium chromate (51Cr) (Amersham Life
Science Corp., Arlington Heights, IL). Radiolabeled
cells were washed once, re-suspended in 5 ml
CP680, Application of Accelerators in Research and Industry: 17th Int'l. Conference, edited by J. L. Duggan and I. L. Morgan
© 2003 American Institute of Physics 0-7354-0149-7/03/$20.00
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complete RPMI 1640, and incubated an additional 1
hour at 37° C. The cells were washed twice, and
diluted to 500 targets per 100 ml of media. Effectors
at constant or variable E:T ratios in a final volume of
100 µl were added first to the wells of 96 well Vbottom plates. An identical volume of targets, except
as noted below, was added to the appropriate wells.
The co-incubation was done in triplicate groups at
37°C in a 5% CO2/air mixture. Effector cells were preincubated with 5E6 F(ab')2 mAbs for 30-60 minutes at
37° C. After 4 hours of incubation, 100 µl of
supernatant was removed and 51Cr radioactivity was
measured in a liquid scintillation counter. Specific
lysis represented as the mean ± SEM was calculated as
follows:
Lung Clearance Assay
Each mouse received 0.5 ml medium containing 5 x
105 cells into a lateral tail vein. Lungs were removed at
different intervals after infusion, and the radioactivity
was measured in a gamma counter (2,3). The results
are expressed as the geometric mean (95% confidence
limits) percentage recovery of injected radioactivity
for groups of 4-6 mice, using the 5 min. lodging value
as 100%.
Imaging Protocol
Control and treated mice were injected I.V, with
In labeled cells (50-200 µCi, 2.5-5 X 106 cells per
mouse). The animals were imaged with a gamma
camera fitted with a medium energy collimator. The
images were acquired every 5 minutes for one-hour
starting at 15-min. post administration of the tracer. In
a separate experiment, 99mTc SESTAMIBI (~60 µCi
per mouse) was injected first to distinguish the
anatomical features and radiolabeled cells were
injected 15 minutes later and the images acquired as
before. The lung regions were identified. Timeactivity curves were generated for the lung regions for
each type of animal (control vs. treated).
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Percent specific lysis = 51Cr cpm [(ER-SR)/(MR-SR)]
x 100,
where ER is the experimental 51Cr release in the
presence of effector cells, SR is the spontaneous 51Cr
release in the presence of medium, and MR is the
maximum 51Cr release in the presence of 1.0% Triton
X-100.
Radiolabeling of YAC-1 Tumor cells
YAC-1 tumor cells were grown in complete RPMI
medium and were diluted to 1-2X 105 cells/ml to force
them into log phase growth the night before the assay.
The cells were harvested, washed, and 1-3 X 107 cells
were first treated with 10-7 M fluoro-deoxyuridine to
inhibit endogenous thymidine synthesis. Twenty
minutes later, the cells were labeled with 25 µCi 5[125I]iodo-2'-deoxyuridine (IUDR) for 90 minutes. The
cells were washed 3 times, and suspended in medium
without serum at 106 cells/0.5 ml.
YAC-1 lymphoma cells (24-25 X 106) were
suspended in 1-2 ml serum free RPMI medium.
Approximately 1 mCi of 111In-oxine (Mallinckrodt)
was added to the cells and incubated at 37° C for 1520 minutes. The labeled cells were spun and washed
twice to remove any free 111In. The cells were then
suspended in the same buffer. The efficiency of
radiolabeling was >90%.
YAC-1 cells (~6x106) were suspended in serum
free complete RPMI medium. 99mTc HMPAO
(Ceretec, Amersham) 1-2 mCi was added to the cells
and incubated for 10 minutes at room temperature. The
cells were washed twice and re-suspended in the same
medium. The efficiency of labeling was 15-20%.
Radiolabeling of the cells with 67Ga was performed
with either oxine or mercaptopyridine (MPO).
Spontaneous release was >50% with Ga-oxine and was
<5% with Ga-MPO. Lung clearance studies were
performed with cells labeled with Ga-MPO.
RESULTS
Lysis of C1498 leukemia cells by effector cells at
varying ratios of effector (E) to target (T) is presented
in Figure 1. Scintigraphic images of the control and
treated mice injected with radiolabeled cells are
presented in Figure 2. The intensity of activity in the
lungs, representing the retention of labeled cells in the
lungs is inversely related to to NK cell function. Lung
clearance of 111In-oxine labeled YAC-1 lymphoma
cells by C57BL/6 mice is presented in Figure 3. Figure
4. shows the lung clearance of 111In-oxine labeled
YAC-1 lymphoma cells by C57BL/6 (control and
treated) mice obtained from the scintigtaphic data from
the time-activity analysis of the regions of interest.
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FIGURE 1. 5E6 anti-LY49 C/I F(ab')2 mAb treatment
of C57BL/6 mouse NK cell stimulates the lysis of
syngeneic EL4 lymphoma cells labeled with 51Cr.
FIGURE 3. Lung clearance of 111In-oxine labeled
YAC-1 lymphoma cells by C57BL/6 mice. Each value
is the geometric mean of groups of 4 mice.
FIGURE 2
Image 1
PI:PC
Image 2
anti NK1.1
FIGURE 4. Lung clearance of 111In-oxine labeled
YAC-1 cells in C57BL/6 mice treated with no agent
(control), PI:PC to stimulate NK cells or anti NK1.1
mAb to deplete NK cells. Note that retention of
labeled cells is inversely related to NK cell function.
Time-activity curves were obtained by analysis of
region of interest data from scintigraphic images.
Image 3
control
Scintigraphic images of C57BL/6 mice injected with
111
In labeled YAC-1 tumor cells. Animals were treated
with PI:PC (Poly Inosinic Poly Cytodylic acid to
stimulate NK activity), anti NK1.1 mAb (monoclonal
antibody to deplete NK cells) or vehicle (control).
Note that retention of labeled cells is inversely related
to NK cell function.
CONCLUSIONS
Our data show that gamma emitting radionuclides
such as 111In, 99mTc or 67Ga can be used to label tumor
cells without affecting their viability. 67Ga-oxine
labeled YAC-1 lymphoma cells showed very high
spontaneous release of radioactivity (>50%), however,
Ga-MPO labeled cells had low (<5%) spontaneous
release, and may be used for lung clearance studies.
The lung clearance of 111In, 99mTc or 67Ga labeled
YAC-1 tumor cells in C57BL/6 mice was very similar
to the cells labeled with 51Cr or 125I-IUDR. These
labels may be used to determine lung clearance by
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scintigarphic imaging techniques. 68Ga obtained from
a generator may be used to label target cells to study
the lung clearance with a small animal PET imaging
system (MICROPET or similar device). In summary:
1. Gamma emitting radionuclides can be used to label
tumor cells for in vivo imaging studies. Tumor cell
viability is not adversely affected by the labeling
procedure.
2. The scintigraphic images accurately reflect the
ability of NK cells to lyse tumor cells in vivo.
3. These agents can be used to evaluate the effect of
blocking negative signals to NK cells that result in
better antitumor function.
ACKNOWLEDGEMENT
The investigations were conducted in conjunction
with the Southwestern In Vivo Cancer Cellular and
Molecular Imaging Center, P20 CA086354
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