-
(CANCER RESEARCH 49. 3742-3746. July 15, 1989]
Low Deformability of Lymphokine-activated Killer Cells as a
Possible Determinantof i/i Vivo Distribution1
Akihiko Sasaki,2 Rakesh K. Jain,3 Azzam A. Maghazachi, Ronald H.
Goldfarb, and Ronald B. Herberman
Tumor Microcirculation Laboratory, Department of Chemical
Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania
15213-3890 ¡A.S., R. K. J.], andPittsburgh Cancer Institute, and
Department of Pathology and Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania 15213-2592 [A. A. M., R. H. G., R. B.
H.J
ABSTRACT
Successful therapy of tumors with lymphokine-activated killer
(LAK)cells is presumably dependent on their cytolytic potential and
theirgaining access to the target cells through the
microcirculation. The latterprocess involves dissemination through
the microvessels, adhesion to thevenular walls, and extravasation
through them, all of which depend onthe size and deformability of
these effector cells. The aim of the presentstudy was to measure
the deformability of these cells quantitatively usinga micropipet
aspiration technique and to analyze the deformation datausing a
mathematical model which yielded parameters indicative of
therigidity of the cell membrane and the cytoplasm. Adherent rat
LAK cells,consisting of a highly purified population of interleukin
2-activated largegranular lymphocytes, with high cytotoxicity were
obtained by a recentlydeveloped method. The deformability
characteristics of fresh large granular lymphocytes (mean diameter,
7.2 um), LAK cells (11.0 urn), freshT-cells (6.6 urn), and
concanavalin A-activated T-cells (9.7 urn) werecompared. LAK cells
were significantly less deformable than other celltypes (about
one-half at an aspiration pressure of —¿�25mm of ILO (/' <
0.001 )|. Cell deformability was independent of cell size and
calciumcontent of the medium. Analysis of the data with the
mathematical modelsuggested that both the cell membrane and the
cytoplasmic factorscontributed to the rigidity of LAK cells. This
increased rigidity coupledwith their large cell size may explain
high retention of LAK cells in thelungs immediately after i.v.
injection and a reduction in tumor targettingdue to external
radiation. Finally, these results suggest that LAK celltherapy
might be enhanced by intraarterial injection into an organ
infiltrated by tumor métastases.
INTRODUCTIONAdoptive immunotherapy with LAK4 cells offers a
novel
approach to the treatment of solid tumors (1-5). The
efficiencyof cancer therapy with LAK cells presumably depends on
bothcytolytic potential of these cells and their localization in
tumortissue. The latter parameter consists of several steps,
includingthe distribution in the body, entrapment at the capillary
entrance, adhesion to venular walls, and extravasation.
Unfortunately, LAK cell therapy is currently limited by the toxic
sideeffects on lung and other normal tissues.
Recent in vivo distribution studies have shown that 2 h
afteri.v. injection, a higher proportion of LAK cells are
entrappedin the rat lungs than are LGL (47% versus 22%).
Similarly,more AT cells are entrapped in the lungs than FT cells
(40%versus 5%) (6-8). In addition to the presumed adhesion of
thesecells to the endothelial and/or cancer cells (9-13), it is
presently
Received 2/8/89; accepted 4/10/89.The costs of publication of
this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
advertisement inaccordance with 18 ll.S.C. Section 17.14solely to
indicate this fact.
1Supported by the National Science Foundation and the American
CancerSociety. This work was presented at the FASEB Meeting, May
2-5, 1988, LasVegas. NV. and at the Radiation Research Society
Meeting. March 20-23. 1989.Seattle, WA.
•¿�Recipient of a fellowship from the Japanese Agency of
Science and Technology) 1987-1988).
1To whom requests for reprints should be addressed.'The
abbreviations used are: LAK, lymphokine-activated killer cell;
LGL,
large granular lymphocyte; AT. concanavalin A-activated T-cell;
FT, fresh T-cell;IL-2, interleukin 2; con A, concanavalin A: FCS.
fetal calf serum; LAL, largeagranular lymphocytes; CM. complete
medium; d(f). deformation with time; Dc.cell diameter; Dp,
micropipet diameter; d(f)/Dp, normalized deformation.
unclear as to what mechanisms are responsible for this
differential entrapment of activated cells. An understanding of
thedeterminants of in vivo localization may suggest approaches
forcontrolling or preventing normal tissue toxicity and, hence,may
lead to improved strategies for adoptive immunotherapyof
cancer.
Cell entrapment in vivo of various cell types is known todepend
strongly on their size and deformability characteristics(14-17).
Since LAK and AT cells have a relatively large diameter, and the
former cells contain numerous cytoplasmic granules, these cells may
have high resistance to flow. The in vivodistribution and in vitro
morphological characteristics of thesecells suggest the following
hypothesis: LAK cells are more rigidthan LGL; and AT cells are more
rigid than FT cells.
To test this hypothesis, the deformability of these cells aswell
as of the WBC was quantitated using a micropipet aspiration
technique (14, 18). In this technique, negative pressure issuddenly
applied to the cell membrane via a micropipet. Theresulting
deformation data are analyzed using an appropriatemathematical
model to obtain viscoelastic coefficients for themembrane and the
cytoplasm (19).
MATERIALS AND METHODS
Cell Preparation. LGL and FT cells were prepared from the
spleensof male Fischer 344 rats (75 to 100 g; Taconic Farms, German
Town,NY). A single cell suspension in RPMI 1640 medium (Gibco)
with10% FCS was centrifugea on a Ficoll-Hypaque (Pharmacia Fine
Chemicals, Uppsala, Sweden) density gradient to get mononuclear
cells.Adherent monocytes, macrophages, and B-cells were depleted by
passage through a nylon-wool column (Cellular Products, Buffalo,
NY).LGL and FT cells were obtained as separate fractions by
centrifugaronon Percoli (Pharmacia) density gradients (20).
Purified LAK ceils were prepared using a recently developed
method(21). Briefly, LGL (2 x 106/rnl) were cultured with human
recombinantIL-2 (1000 units/ml; Cetus Co., Emeryville, CA) in 5%
CO2/95% airat 37°Cin a CM consisting of RPM1-1640, 10% FCS, 2 mi\i
glutamine,5 x 10"' M 2-mercaptoethanol, 1% minimal essential
medium, nones-
sential amino acids, and antibiotics. After 2 days, 94 to 98% of
IL-2-induced adherent cells had morphological and phenotypical
characteristics of LGL and showed very high LAK activity in a
standard 4-h5lCr release assay. They were cultured for an
additional 3 days with thefiltered conditioned medium (0.2-um
Nalgene filter). Adherent LAKcells were removed from the culture
flask surface in phosphate-bufferedsaline with 5 mi\t EDTA, washed
with CM without IL-2, and stored uptol2hat4°C.
AT cells were obtained from FT cells (5 x I06/ml) by culturing
with
5 ¿ig/mlof Con A (Sigma) for 3 days (22). Contaminating LGL
wereremoved by incubation with 40 mM i.-leucine-methylester (Sigma)
for30 min at 37°Cfollowed by Ficoll-Hypaque gradient
centrifugation (8).
WBC were obtained by centrifuging rabbit peripheral blood
accordingto the procedure described in Ref. 19.
Experimental System. The experimental system is similar to the
onepreviously used in our laboratory ( 18). Briefly, an inverted
microscope(Diaphot; Nikon) was used for white light illumination,
with heat-absorbing filters, a Turret condenser (numerical
aperture, 0.52; workingdistance, 20.5 mm), a lOOx oil immersion
objective, and a 20x projection lens connected to a television
camera (ITC-400; Ikegami). A video
3742
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LAK CELL DEFORMABILITY
cassette recorder (IAG-6500; Panasonic) with a time code
generator(WJ-810; Panasonic) and a television monitor (PM-205A;
Ikegami)were used to record and display the images.
The pressure application system consisted of a damping chamber,
athree-way manifold, two water reservoir bottles, and connecting
tubes.The damping chamber partially filled with saline was adjusted
to givea slight negative pressure (
-
LAK CELL DEFORMABILITY
200
0.4
0.3
0aX 0.2
0.1
0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2
TIME (SEC)
aO
0.4
0.3
0.2
0.1
0.0
CM + EDTA
0.0 0.2 0.4 0.6 0.8 1.0 1.2
TIME (SEC)Fig. 2. Time course of normalized deformation,
d(f)/Dp, of LAK, LGL, AT,
FT cells, and rabbit WBC in CM (A) and CM + EDTA (B). Points,
mean; bars,SEM; numbers in parentheses, number of cells.
Significant differences betweencell types shown by specific marks
are: *, LAK versus LGL, AT, FT, and WBCin both CM and CM + EDTA
(0.03 to 1.0 s, P < 0.001 ); t, WBC versus LGL inboth CM and CM
-I-EDTA (0.1 and 0.3 s, /> < 0.05), WBC venus AT in CM(0.3 to
1.0 s), and in CM + EDTA (0.1 to 1.0 s, P< 0.05); t, FT and AT
in bothCM and CM + EDTA (0.3 to 1.0 s, P < 0.05). No significant
differences betweenmedia were observed except for WBC at 0.3 to 1.0
s (P < 0.05).
Quantitation. LAK cells were about twice as rigid as LGL,AT
cells, or FT cells (P < 0.001). AT cells were also more
rigidthan FT cells (P < 0.05). There was no significant
differencebetween CM and CM + EDTA samples in either cell
group.Correlation between either d(0.1)/Dp or d(1.0)/Dp and the
celldiameter was not significant except for d(1.0)/Dp of LAK
cellsin CM (r = 0.31, P = 0.05). Two-dimensional distribution
ofeach cell group by cell diameter and d(1.0)/Dp showed
rheolog-ical subpopulations of LAK and AT cells which were
distinctfrom other subpopulations of LGL, FT cells, and WBC.
Therefore, the product of Dc and [d(f)/Dp]"', hereafter referred to
as
a resistance factor, was compared (Fig. 3). The value for
LAKcells was 3-fold as large as for LGL and FT cells (P <
0.001),and the value for AT cells was 1.5 times larger than that
forFT cells (P < 0.01). All these cells, in turn, were more
rigidthan WBC.
Viscoelastic coefficients gave results similar to d(r)/D,,
(Fig.
LAK LGL AT FT WBC
CELL TYPES
Fig. 3. Resistance factor [D0 Dp/d(1.0); mean ±SE] for LAK,
LGL, AT, FT,and WBC in CM and CM + EDTA. Statistical comparisons by
unpaired Student's/ test are shown between cell types and media (*,
P < 0.001; t, P < 0.01; t. P <0.05). Note that LAK cells
would offer more resistance to deformations inmicrovessels than
would other cell types.
4; Table 1). LAK cells have approximately twice as large k,,
k2,and n as LGL, AT cells, or FT cells. AT cells had
significantlylarger k, and ¿ithan LGL or FT cells in CM. The
differencebetween cells in CM and CM + EDTA was only significant
ink, of FT cells. The ratio of viscoelastic coefficients
betweenfresh and activated cells (LAK/LGL and AT/FT) showed
adifferent effect of activation on the cell structure. LAK/LGLhad a
ratio of about 2 in all viscoelastic coefficients, but AT/FT had
the ratio of less than 1.5 (Table 1). These resultsdemonstrate that
LAK cells have increased values of k,, k2, andM,but AT cells have
increased values of only k, and ¡i.
DISCUSSION
The objective of the present study was to measure the
deform-ability of LAK cells in vitro and to evaluate its
implications fortheir distribution in vivo. Deformability is
dependent on multiple parameters, including cellular morphology,
cytoskeletonstructure, IL-2 concentration, discharge of cytoplasmic
granules, and Ca2+ content. LAK cells were found to be about
half
as deformable as LGL and FT cells. AT cells were less
deformatale than FT cells, but were not as rigid as LAK cells (Fig.
2).The viscoelastic coefficients of LAK cells showed that both
thecell membrane and the cytoplasm contributed to the
decreaseddeformability (Table 1). These results on the increased
rigidityof LAK and AT cells are in agreement with the hypothesis
thatthe initial cell entrapment in the lung after i.v. injection
isdetermined by cell rigidity and diameter (15, 16).
WBC from rabbit peripheral blood were more deformablethan any
other cell types except for FT cells. The viscoelasticcoefficients
of WBC at -5 mm of H2O aspiration pressure had
values similar to those of human peripheral blood measured ata
lower pressure range (—2.8to 9.0 mm of H2O) (14). However,when
measured at -25 mm of H2O aspiration pressure, the
value of viscoelastic coefficients of rabbit WBC was higher
thanthat measured at —¿�5mm of H2O.
In contrast to the homogeneous appearance of LGL, LAKcells were
heterogeneous in cell size and the content of cytoplasmic granules,
presumably due to difference in cell maturation and stages in the
cell cycle. Two morphological types ofhuman LAK cells have been
described, which vary in themorphology of their cytoplasmic
granules (23, 24). In thepresent study with rat adherent LAK cells,
we occasionally(
-
LAK CELL DEFORMABILITY
h, 4515 dyn/cm2k2 6068 dyn/cm2., 2226 dyn s/cm2
Table 1 I'iscoelastic coefficientsStatistical significance was
by Student's unpaired / test.
Time constantCell types k, (dyn/cm2) k2(dyn/cm2) ? (dyn/s/cm2)
(s)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
TIME (SEC)
k, 2179 dyn/cm2k2 3332 dyn/cm2u 1190 dyn s/cm2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
TIME (SEC)Fig. 4. Simulation of the cell deformability by
standard viscoelastic model and
the viscoelastic coefficients (ki, kj, and ji) of LAK and LGL
(A) and of AT andFT cells (A). For details of the model, see the
text.
cytoplasmic granules. In separate experiments, preparations
ofcells were depleted of LGL by L-leucine methylester resultingin a
pure population of cells that are precursors to natural killercells
(LAL) (25). LAL were compared with normal LGL, butno significant
difference in the deformability was observedbetween them.5 We did
not observe any changes in cellular
integrity of morphologically normal cells due to aspiration
at-25 mm of H2O except for the occasional small tear of the
cell
membrane at the deformed cap.The diameter and deformability of
LAK cells were poorly
correlated in spite of diverse distribution of the cell
diameterdue to growth and differentiation. This observation
suggeststhat it may not be possible to select LAK cells a priori
withspecific deformability on the basis of cell size. In addition,
acorrelation was not observed between viscoelastic coefficientsand
cell diameter in either cell group suspended in CM or CM+ EDTA. It
seems likely that the in vivo ability of LAK cells topass through
capillaries depends on their deformability as well
CMLAK(41)"LGL
(58)AT(45)FT(49)WBC
(19)CM
+EDTALAK(63)LGL(34)AT(31)FT*(42)WBC
(27)67132637±^3878
±2509¿2004
±71193113350831141582++±±±433*'
rMS'-'238^138'159312*234^247f159^96.179383888±±4346
±3829±2793
±85783774364337752834±±±±-4-654*190^244^175^336571*252'284'2202262733135316631215±¿++952±3195136814421397839+-t±204*71.1'95.
y51.6a193.0210*Vt.tf±
89./±51.60.764
±0.892±0.852±0.839±0.847±0.862
±0.852±0.849±0.842
±0.0
17*'0.020'0.0230.0190.0420.028'0.0320.0310.0250.876
±0.026°Numbers in parentheses, number of cases.* LAK versus
LGL. AT. FT. and WBC (P < 0.001 ).c Mean ±SE.* P
-
LAK CELL DEFORMAB1LITY
from capillaries in a manner similar to neutrophils' (33).
The tumor microvessels have sluggish blood flow;
dilated,saccular, and partially collapsed vessels; and poorly
developedbasement membrane ( 17). Therefore, after the LAK cells
enterthe tumor microcirculation, their adhesion and
extravasationmay be much easier in tumors compared to several
normaltissues. Unfortunately, following the conventional systemic
injection, very few LAK cells arrive at the sites of tumor
growthand, hence, a relatively small fraction of total cells
injectedlocalizes in tumors (2). A decrease in tumor interstitial
pressurecaused by radiation may open up partially collapsed
tumorvessels (34) and may further reduce entrapment of LAK
cellsand/or facilitate escape of entrapped LAK cells.
Therefore,external beam radiation may lead to a reduction in uptake
ofLAK cells by tumors (35). One possible method of increasingthe
uptake of LAK cells by a tumor would be the local arterialinjection
of LAK cells in the organ infiltrated by tumor métastases. This
approach would bypass the initial entrapment ofLAK cells in other
normal organs (e.g., lungs, liver, and spleen)and might take
advantage of the peculiar nature of the tumormicrocirculation (17).
This hypothesis remains to be tested byintraarterial injection of
LAK cells and by direct observation oftheir behavior in the normal
and tumor microcirculation (16).
ACKNOWLEDGMENTS
We thank Dr. Shu Chien, Dr. Geert W. Schmid-Schoenbein,Dr. K.-L.
P. Sung, Dr. Paul Frattini, Dr. Theresa L. Whiteside,and Dr. Robert
J. Melder for helpful suggestions; and Caryn Giffen,Peter Lindholm,
G. Ray Martin, Michelle Clauss, and Dr. RichardStock for technical
help.
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Research. cancerres.aacrjournals.org Downloaded from
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1989;49:3742-3746. Cancer Res Akihiko Sasaki, Rakesh K. Jain,
Azzam A. Maghazachi, et al.
Distributionin VivoPossible Determinant of Low Deformability of
Lymphokine-activated Killer Cells as a
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