(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.

REFERENCES

1. Rosenberg, S. A., Lotze, M. T., Leitman, S., Chang, A. E., Velio. J. T..Seipp. C. A., and Simpson, C. A new approach to the therapy of cancer basedon the systemic administration of autologous lymphokine-activated killercells and recombinanl inlerleukin 2. Surgery (St. Louis), S: 262-270, 1986.

2. Maghazachi. A. A., Goldfarb. R. H., Krislon. R. T., Hiserodt, J. C., Giffen.C. A., and Herberman. R. B. In rivo distribution of interleukin 2 activatedcells. In: E. Lotzova and R. B. Herberman (eds.), IL-2 Activated Killer Cells.Boca Raton, FL: CRC Press, in press, 1989.

3. Phillips. J. H., and Lanier, L. L. Dissection of the lymphokine-activatedkiller phenomenon. Relative contributions of peripheral blood natural killercells and T-lymphocytes for cytolysis. J. Exp. Med., 164: 814-825, 1986.

4. Herberman, R. B., Hiserodt, J. C., Vujanovic, N., Balch. C., Lotzova, E.,Bolhuis. R., Golub, S., Lanbier, L. L.. Phillips. J. H.. Riccardi. C., Ritz, J.,Santoni, A., Schmidt, R. E., and Uchida, A. Lymphokine-activated killer cellactivity. Characteristics of effector cells and their progenitors in blood andspleen. Immunol. Today, 8: 178-181, 1987.

5. Hiserodl, J. C., Vujanovic. N. L., Reynolds, C. W., Herberman, R. B., andCramer, D. V. Lymphokine activated killer cells in rats: analysis of precursorand effector cell phenotypes and relationship to natural killer cells. In: E.Truitt, R. P. Gale, and M. Borden (eds.). Cellular Immunotherapy of Cancer,pp. 137-146. New York: Alan R. Liss, 1987.

6. Reynolds. C. W., Denn, A. C., Ill, Barlozzari, T.. Wiltrout, R. H., Reichardt.D. A., and Herberman. R. B. Natural killer activity in the rat. IV. Distributionof large granular lymphocytes (LGL) following intravenous and intraperito-neal transfer. Cell. Immunol., 86: 371-380, 1984.

7. Rolstad, B., Herberman, R. B., and Reynolds, C. W. Natural killer cellactivity in the rat. V. The circulation patterns and tissue localization ofperipheral blood large granular lymphocytes (LGL). J. Immunol., 136:2800-2808, 1986.

8. Maghazachi. A. A., Herberman, R. B., Vujanovic, N. L.. and Hiserodt, J. C.In vivo distribution and tissue localization of highly purified rat lymphokineactivated killer (LAK) cells. Cell. Immunol., 115: 179-194, 1988.

9. Argov, S., Hebdon, M., Cuatrecasas. P., and Koren, H. S. Phorbol ester-induced lymphocyte adherence: selective action of NK cells. J. Immunol..134: 2215-2222, 1985.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Goldfarb, R. H. Cell-mediated cytotoxic reactions. Hum. Pathol., 17: 138-145. 1986.Roozemond, R. C., Mevissen. M., Urli, D. C., and Bonavida, B. Effect ofaltered membrane structure on NK cell-mediated cytotoxicity. III. Decreasedsusceptibility to natural killer cytotoxic factor (NKCF) and suppression ofNKCF release by membrane rigidification. J. Immunol., 139: 1739-1746,1987.Foa, C., Mege, J.-L., Capo. C., Benoliel, A.-M., Galindo, J.-R.. and Bon-grand, P. T-cell-mediated cytolysis: analysis of killer and target cell deformabili!)' and deformation during conjugate formation. J. Cell Sci., 89: 561-573, 1988.Aronson, F. R., Libby. P. L.. Brandon. E. P., Janicka, M. P., and Mier, J.W. IL-2 rapidly induces natural killer cell adhesion to human endothelialcells: a potential mechanism for endothelial injury'- •¿�'•Immunol., / //: 158-163, 1988.Chien, S., Schmid-Schoenbein, G. W., Sung. K.-L. P., Schmalzer, E. A., andSkalak, R. Viscoelastic properties of leukocytes. In: H. J. Meiselman, M. A.Lichtman, and P. L. LaCelle (eds.). White Cell Mechanics: Basic Scienceand Clinical Aspects, pp. 19-51. New York: Alan R. Liss, 1984.Simchon, S., Jan, K.-M., and Chien, S. Influence of reduced red cell deform-ability on regional blood How. Am J. Physiol., 253: H898-H903, 1987.Jain. R. K., and Ward-Hartley, K. A. Dynamics of cancer cell interactionswith microvasculature and interstitium. Biorheology, 24: 117-125, 1987.Jain. R. K. Determinants of tumor blood flow: a review. Cancer Res., 48:2641-2658, 1988.Traykov, T. T., and Jain. R. K. Effect of glucose and galactose on red bloodcell membrane deformability. Int. J. Microcirc. Clin. Exp., 6: 35-44, 1987.Schmid-Schoenbein, G. W., Sung, K.-L. P., Tozeren. H., Skalak, R., andChien, S. Passive mechanical properties of human leukocytes. Biophys. J.,36: 243-256, 1981.Reynolds, C. W., Timonen, T., and Herberman, R. B. Natural killer cellactivity in the rat. I. Isolation and characterization of the effector cells. J.Immunol., 127: 282-287, 1981.Vujanovic, N. L., Herberman, R. B., Maghazachi, A. A., and Hiserodt, J. C.Lymphokine-activated killer cells in rats. III. A simple method for thepurification of large granular lymphocytes and their rapid expansion andconversion into lymphokine-activated killer cells. J. Exp. Med., 767: 15-29,1988.Maghazachi, A. A., and Philips-Quagliata, J. M. Con A-propagated, auto-reactive T-cell clones that secrete factors promoting high IgA responses. Int.Arch. Allergy Appi. Immunol.. 86: 147-156, 1988.Ferrarini, M., and Grossi, C. E. Ultrastructure and cytochemistry of thehuman large granular lymphocytes. In: E. Lotzova and R. B. Herberman(eds.). Immunology of Natural Killer Cells, Vol. 1, pp. 33-43. Boca Raton,FL: CRC Press, 1986.Zarcone, D.. Prasthofer, E. F., Malavasi, F., Pistoia, V.. LoBuglio, A. F.,and Grossi, C. E. Ultrastructural analysis of human natural killer cellactivation. Blood, 69: 1725-1736, 1987.Maghazachi, A. A., Herberman, R. G., Vujanovic, N. L., and Hiserodt, J. C.Lymphokine-activated killer cells in rats. IV. Developmental relationshipsamong large agranular lymphocytes (LAL), large granular lymphocytes(LGL), and lymphokine-activated killer (LAK) cells. J. Immunol., 140:2846-2852, 1988.Schmid-Schoenbein, G. W., Skalak, R., Sung, K.-L. P., and Chien, S. Humanleukocytes in the active state. In: U. Bagge, G. V. R. Born, and P. Gaehtgens(eds.). White Blood Cell, pp. 21-31. The Hague: Martinus Nijhoff Publisher,1982.Sung, K.-L. P., Schmid-Schoenbein, G. W. Schuessler, G. B., and Chien, S.Rheological properties of T- and B-lymphocytes and lymphoblastoid cells(abstract). Biorheology, 23: 284. 1986.Lichtman, M. A. Rheology of leukocytes, leukocyte suspensions, and bloodin leukemia: possible relationship to clinical manifestations. J. Clin. Invest.,52:350-358, 1973.LaCelle, E. P. L., Bush, R. W., and Smith, B. D. Viscoelastic properties ofnormal and pathologic human granulocytes and lymphocytes. /;;.-U. Bagge,G. V. R. Born, and P. Gaehtgens (eds.). White Blood Cell, pp. 38-45. TheHague: Martinus Nijhoff Publisher, 1982.Wood, S. Pathogenesis of metastasis formation observed in vivo in the rabbitear chamber. Arch. Pathol., 66: 550-568, 1958.Zeidman, 1. The fate of circulating tumor cells. I. Passage of cells throughcapillaries. Cancer Res., 21: 38-39, 1961.Herberman, R. B., Melder, R. J., Walker, E., and Whiteside, T. L. The roleof p 150,95 antigen of human IL-2 activated natural killer (NK) cells to solidsurfaces (abstract). FASEB J., 2: A445, 1988.Branemark, P.-I., and Lindstrom, J. Shape of circulating blood corpuscles.Biorheology, /: 139-142, 1963.Jain, R. K., and Baxter, L. T. Mechanism of heterogenous distribution ofmonoclonal antibodies and other macromolecules in tumors: significance ofelevated interstitial pressure. Cancer Res., 48: 7022-7032, 1988.Munshi, N. C., and Williams. J. R. Effect of external beam irradiation onLAK cell targeting (abstract Dl-19). In: Proceedings of the Radiation Research Society, 36th Annual Meeting, Philadelphia, April 16-21, 1988.

3746

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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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