Transplantation of Human Hairy Cell Leukemia in Radiation-Preconditioned Nude Mice: Characterization of the Model by Histological, Histochemical, Phenotypic, and Tumor Kinetic Studies

Blood, Vol 71, No 6 (June), 1988: pp 151 1-1517 1511

B L 0 OD The American

The JSociety

ournal of

of Hematology

VOL 71, NO 6 JUNE 1988

Transplantation of Human Hairy Cell Leukemia in Radiation-PreconditionedNude Mice: Characterization of the Model by Histological, Histochemical,

Phenotypic, and Tumor Kinetic Studies

By Guy B. Faguet and Julia F. Agee

Two cell lines (EH and HK) with hairy cell leukemia (HCL)

immunophenotypes were recently derived from two HCL

patients. Both cell lines were transplanted subcutaneously

(2 x 10 or 2 x 10/mouse) in male BALB/c nu/nu mice

(n = 1 28) with a 97% success rate when coimplanted with

nonproliferative HT-1080 fibrosarcoma cells (2 x 101

mouse) in recipients preconditioned with total-body irra-

diation (200 R weekly for 3 weeks). Tumors appeared five

to ten days postimplant and reached up to 25% of body

weight after a mean survival of 8 weeks (range, 30 to 90

days). Tumor histology suggested large cell lymphoma.

Cytochemically and immunophenotypically. tumor cells

were indistinguishable from their parent cells. Species and

A NUMBER of human solid neoplasms are readilytransplantable in athymic mice.’6 These xenotrans-

plantation models provide in vivo environments for the initial

assessment of novel approaches to chemotherapy, radiother-

apy, or immunotherapy7�2 that are not ethically permissible

in the human. These models also enable the study of metas-

tases by using probes such as radiolabeled monoclonal anti-

bodies (MoAbs)’3’5; however, the observation that xeno-

grafted tumors are often less metastati&6”7 than the parent

human neoplasms and that leukemia transplantation

requires further immunosuppression ofthe host’822 limits the

usefulness of xenotransplantation models. The increased

natural killer (NK) cell activity23’24 and antitumor antibodies

naturally occurring in nude mice25 have been postulated to

play a role in the rejection of leukemia implants. An addi-

tional factor might be the tendency of leukemia cells to

infiltrate local tissues rather than to form nodules that

further limit implantation. Such behavior might relate to a

lack of growth-, angiogenesis-, or other implantation-

promoting factors)7’26

In this report we present our experience with transplanta-

tion of human hairy cell leukemia (HCL) cell lines developedin our laboratory27 in radiation-preconditioned, congenitally

athymic mice. The transplantation yield was nearly 100%.

Tumors grew locally and metastasized extensively to lym-

phoid and extralymphoid tissues. This xenotransplantation

model might be valuable for assessing the potential useful-

ness of new approaches for the treatment of HCL.

lineage derivation of tumor cells was confirmed by antibody

probes against the mouse histocompatibility antigen H-2,

human T and B lymphocyte antigens, and the HCL-

associated common chronic lymphocytic leukemia antigen

(cCLLa). In order of decreasing frequency, metastases

occurred in the spleen, lungs. pleura. lymph nodes. bone

marrow. and kidneys. Up to 12% of circulating lymphoid

cells in mice were cCLLa-positive. which suggested hema-

togenous tumor dissemination. This HCL xenotransplanta-

tion model might be useful in preclinical studies for

exploring novel experimental therapies for the manage-

ment of human HCL.

S 1988 by Grune & Stratton, Inc.

Animals

MATERIALS AND METHODS

Athymic nude mice, BALB/c (nu/nu) male, 4 to 6 weeks of age,

and weighing 20 to 35 g, were supplied by the Office of MammalianGenetics and Animal Production, National Cancer Institute, Fred-crick Cancer Research Center, Frederick, MD. The mice were kept

under sterile conditions in cages with fiberglass hoods in a roomequipped with a negative air flow system and were fed a sterilizedmouse diet and sterile water. All procedures were performed under

strict sterile conditions.

Cell Lines

Molt-4,28 a human T cell line, and HT-1080,29 a fibrosarcoma cellline, were supplied by the American Type Culture Collection,

Rockville, MD, and grown in RPMI 1640 medium (GIBCO, Grand

From the Medical Research Services, Veterans Administration

Medical Center, Augusta, GA. and Departments of Medicine and

Cell and Molecular Biology, Medical College of Georgia.

Submitted June 26, 1987; accepted January 13, 1988.

Address reprint requests to Guy B. Faguet. MD, Veterans

Administration Medical Center (I I IN). Augusta, GA 30912.

The publication costs ofthis article were defrayed in part by page

charge payment. This article must therefore be hereby marked

“advertisement” in accordance with 18 U.S.C. section 1 734 solely to

indicate this fact.

©I 988 by Grune & Stratton, Inc.

0006-4971/88/7106-0026$3.00/0

1512 FAGUET AND AGEE

Island, NY) supplemented with 10% fetal calf serum (FCS) as

suspension and monolayer cultures, respectively. Hairy cell lines EH

and HK, developed in our laboratory27 from peripheral blood fromtwo patients with HCL, were grown as suspension cultures in a

medium (Flow Laboratories, Inglewood, CA) supplemented with15% FCS. All cultures were propagated in 75-cm2 tissue culture

flasks (Corning Glass Works, Corning, NY) in a 5% CO2, 95%

humidity incubator (Forma Scientific, Marietta, OH) with mediumreplacement twice weekly. Seeds were stored in liquid nitrogen.

Immunofluorescence Assays

Surface immunoglobulins (SIgs) were assessed by a direct immu-nofluorescence assay using fluorescein isothiocyanate(FITC)-con-

jugated antisera specific for each heavy and light chain. Othersurface determinants were assessed by indirect immunofluorescence(IFA)�#{176}32 by using FI�TC-conjugated goat antimouse Igs (Cappel

Laboratories, Cochranville, PA) and a panel of mouse antihumanMoAbs: OKT series (Ortho Diagnostics, Raritan, NJ); LEU series

(Becton Dickinson, Mountain View, CA); My4, Bi, MO2, and iS

(Coulter Immunology, Hialeah, FL); aHC1 and aHC2 (a gift fromDr D.N. Posnett, The Rockefeller University, New York); and

CLL- 1 and CLL-2,3’ antibodies specific against the common chronic

lymphocytic leukemia antigen (cCLLa), an antigen common to

HCL and related disorders.3’ Antimouse MoAbs Thyl .2 and H-

2Dk/H-2Kk/H-2Dd (Organon Teknika, Irving, TX) were also

used. Antibodies were used in concentrations sufficient to achieve

saturation binding. Cell fluorescence was determined by fluores-cence microscopy on a Leitz epifluorescence microscope (E. Leitz,Inc, Rockleigh, NY). Nonimmune mouse and rabbit antisera and

irrelevant antibodies and target cells were included as negative

controls.

Radiation

After a 2-week observation period to ensure health and acclimati-zation, mice to be irradiated were anesthetized lightly with anintraperitoneal (IP) injection of ketamine (35 �ag/g body weight)placed in 50-mL uncapped sterile plastic tubes covered with sterile

gauze for ventilation before they received three weekly doses of 200R total-body irradiation (TBI). TBI was administered by a thera-

peutic x-ray unit (courtesy of S. Reichard, Medical College of

Georgia, Augusta, GA) operated at 200 kV and 15 mA with a filter

of 0.5 mm Cu and 1 mm Al at dose rates of 40 to 100 R/min. The

half-value was equivalent to 0.9 mm Cu. The dose delivered to thecenter of the body area in roentgens per minute in air was measured

by a Victoreen dosimeter.

Transplantation

A series of pilot studies was first undertaken to assess the

feasibility of transplanting EH and HK cell lines. The transplanta-

tion protocol followed published guidelines including the use of

preirradiated nude mic&82’ and transplantation-promoting irra-diated HT-1080 fibrosarcoma cell lines as coimplants.18 This proto-

col confirmed the successful transplantation of leukemia-derivedMolt-4 cell lines when coimplanted with HT-1080 cells)8 Thus,Molt-4 cells coimplanted with HT-1080 cells were used as trans-plantable controls. Variables studied included cell line origin, route

of administration, size of inoculum, and the need for irradiationpreconditioning of recipient mice and for coimplanting transplanta-tion-promoting HT-l080 cells. Three days postirradiation or at

equivalent times in unirradiated animals, mice were given a singlesubcutaneous (SC) or intraperitoneal (IP) injection of 2 x I 0s or 2 x

106 cells from leukemic cell lines EH, 1-1K, or Molt-4 in the log phase

of growth that were admixed or not with 2 x 106 preirradiated

(5,000 rad) HT-1080 cells in 0.2 mL of RPMI 1640. Cell irradiationwas carried out in an x-ray unit operated at 90 kV and 10 mA at

doses of 1,000 R/min. After inoculation, SC tumor growth was

monitored from three-dimensional diameter measurements obtainedtwice weekly with vernier calipers. SC tumor volume was calculatedaccording to the following formula’: volume = [(4 x 3.14/3) x(L/2) x (W/2) x (D)]/2. IP tumor presence and growth were

assessed by abdominal palpation. SC tumor weights were measuredat autopsy. Blood samples were taken periodically from the tail veinand at autopsy from a heart puncture to monitor blood counts andthe circulation of tumor cells.

Tumor Histology and Immunophenotype

At various times after cell inoculation, mice were killed and

thorough autopsies performed. Tumors were dissected, weighed, andsectioned to assess histology. Tissue samples from tumors and allinternal organs were fixed in 4% formaldehyde fixative, embedded in

paraffin, stained with hematoxylin and eosin, and examined by lightmicroscopy. Cell suspensions were prepared from teased fresh tumorspecimens for assessing cytochemistry and immunophenotype.

Table 1 . Effect o f TBI. HT-1 08 0 Coimplantati on. and Route of Administrat ion on Tra nsplantatio n of HCL ccl I Lines in Nu de Mice

Exp n

cells

HT-1080 TBI Route

Tumors

HK EH MOIT-4 Primary Mets BlOOd

1 6 2 x 10#{176} 0 0 SC 0/6 ND ND

2 6 2 x 1O� 0 600 SC 3/6 ND ND

3 10 2 x 10#{176} 2 x 1O� 600 SC 10/10 ND ND

4 6 2 x 106 0 0 IP 1/6 ND 1/1

5 6 2 x 106 0 600 P 4/6 ND 3/3

6 6 2 x 106 2 x 106 0 P 4/6 ND 4/4

7 10 2 x 106 2 x 106 600 IP 10/10 ND 10/10

8 6 2 x 10� 2 x 106 600 SC 6/6 ND ND

9-10 33 2 x i0� 2 x 10� 600 SC 32/33 11/15 10/10

11-13 95 2 x i0� 2 x 10#{176} 600 SC 92/95 25/32 20/20

Several groups of nude mice (preirradiated or not) received SQ or IP transplants of HK, EH, or Molt-4 cells admixed or not with HT-1080 cells. Mice

were then monitored for the yield and size of primary tumors and the presence and site of metastases. Other studies including blood counts, detection of

circulating malignant cells, and histological and immunophenotypic characterization of tumors were also done as described in the text. Results are

presented as fractions of mice exhibiting SQ or IP tumors (Primary), metastases (Mets), and circulating malignant cells (Blood) in each experiment (Exp)

(ND. not done).

RESULTS

Transplantability ofHCL Cell Lines

Preliminary experiments showed that SC or IP implanta-

tion of 2 x 106 Molt-4 (n = 12) or HK cells (n = 1 2) alone in

irradiated (n = 6) and nonirradiated mice (n = 6) resulted in

erratic or no tumor development, respectively (Table 1).

However, SC and IP transplantability of either cell line inirradiated mice was enhanced by coimplanting irradiation-

treated HT-1080 cells’8: HK cells were inoculated in miceexhibiting pristane-induced ascites to mimic the in vivogrowth environment optimal for hybridomas. This approachyielded abdominal tumors with diffuse peritoneal and mesen-

teric seeding in 68% of the animals (n = 28). In these mice,

up to 75% (mean, 36.3%; SEM, 8.4%) ofthe free ascitic fluid

cells expressed the characteristic hairy cell immunopheno-type (Table 2); likewise, 97% of 128 preirradiated mice

inoculated SC with either EH (n = 33) or HK (n = 95) cells

and 100% of ten control mice receiving Molt-4 cells devel-

oped SC tumors when coimplanted with irradiated HT-1080

cells. When this transplantation protocol was used, the two

EE

K

Lu

-a0>0

I-A

I-IC,Lu

OBSERVATION (DAYS)

10

5 0�

0 5

B VOLUME lx 10” mm’)no

14

Table 2. Immunophenotype in HCL

Markers

HCL Lines

EH HK

Tce)l

OKT3 1 0

OKT4 0 1

OKT6 0 0

OKT8 1 0

LEU-1 0 1

B cell

IgA 0 0

lgD 0 0.5

gE 0.5 0.5

IgG 58 0

gM 1 61

Poly-V 6 1 62

K 59 62

A 0.5 1

Bi 52 48

B2 4 33

LEU-12 29 41

LEU-14 39 31

Myeloid

LEU-M4 0 0

Monocyte

My-4 0.5 1

M0-2 0.5 0

LEU-M5 47 22

Miscellanea

J-5 0 0

aHC1 14 22

aHC2 23 33

cCLLa

aCLL 48 39

CLL-1 49 38

CLL-2 49 39

28 42 56C�

SURVIVAL (DAYS)

70 84

The immunophenotypes of HCL cell lines EH and HK were assessed by

IFA. Results shown represent percent reactive cells from at least 600

cells examined per assay.

different sized inocula assessed (2 x l0� and 2 x 106) were

equally effective in inducing tumors. Thus, all subsequent

experiments were done by SC inoculation of 2 x 10� cells andincluded coimplants. The intravenous route was not assessed.The latent period between SC implantation and the firstevidence of tumor growth was usually seven days (Figure

1A); however, in some cases tumors were palpated as early as

four to five days or as late as ten to 1 2 days. SC tumors werealso successfully transplanted to first-generation preirra-

XENOTRANSPLANTATION OF HCL 1513

Fig 1 . (A) Tumor growth in mice (n 20) bearing HK tumors.HK cells (2 x 1 0) were implanted (along with 2 x 1 0’ nonprolifer-ative HT-1 080 cells) SC in preirradiated mice. At the timesindicated. three-dimensional tumor diameters were measured.and tumor volumes were calculated according to formuladescribed in the text. Tumor volumes (mm3) are expressed as afunction of time (days). (B) Correlation between calculated tumorvolumes and actual tumor weights. At various times postimplant.tumor volumes in mice (n - 20) bearing HK tumors were calcu-lated from three-dimensional diameter measurements. Mice werethen killed and tumor weights recorded. Tumor weights (grams)are expressed as a function of volumes (mm3). (C) Survival of micebearing HK tumors. The survival of mice depicted in panel A isshown as a percentage of the group and is expressed as a functionof time (days).

1514 FAGUET AND AGEE

diated mice (n = 6) without co-implants.No attempts were

made to assess the serial transplantation potential of SC

tumors or the minimum number of cells necessary for

implantation, and no correlation trends were seen between

inoculum size and tumor growth rates, metastases, or host

survival. No appreciable differences were observed between

EH and HK cell lines in terms of the implantation or growthrates of primary tumors or their ultimate size, development,

distribution or size of metastases, tumor histology, or host

survival.

Characterization of Tumors

Tumor growth. Tumor growth was initially slow butproceeded uninterruptedly until death. In one representative

experiment involving 20 mice bearing SC HK tumors, the

following observations were made: (a) there was an excellent

correlation (r = .96) between calculated tumor volume and

actual tumor weight measured at autopsy (Fig 1B). The

volume-weight ratio was I mm3/ I mg; (b) tumors grew to an

average calculated weight of approximately 2 and 4 g at 40

and 60 days postimplant, respectively (Fig 1A), and reached

an average weight of 5.09 g (SEM, I .7) after a mean survivalof 56 (SEM, 18) days; (c) attrition, appeared to nonran-

domly affect mice with larger tumors first, began in thesecond month postimplant and continued at a constant rateof approximately 12% per week (Fig I C); (d) the total body

weight at the end of each experiment showed only a slight

gain, which suggested that normal tissue weight loss approxi-

mately equivalent to that of the growing tumors had taken

place. Thus, after 30 to 45 days posttransplant, most animals

were cachectic, having lost 1 0% to I 5% of their initial body

weight in spite of adequate dietary intake.

Tumor dissemination. Primary tumors disseminated

extensively to lymphoid and nonlymphoid organs. Local

dissemination occurred by contiguous invasion of SC tissue

including fat and muscle. Diffuse infiltration of spleens with

splenomegaly was universal. Some but not all animals exhib-

ited nodal infiltrations as well. Distant micrometastases andmacrometastases of internal organs were identifiable in 77%

of mice in the following decreasing order of frequency:

spleen, lungs (Fig 2A), pleura lymph nodes (Figure 2B),

bone marrow, liver, and kidneys. Brains were not examined.

Blood obtained at autopsy in mice bearing SC EH tumors

(n = 10) and IP HK tumors (n = 18) demonstrated that 0%

to 12% and 5% to 75% of circulating mononuclear cells

exhibited the characteristic hairy cell immunophenotype,

respectively. Circulating tumor cells were evident in many

tissue sections, particularly the lung (Fig 3C).

Histology, histochemistry, and cytology. To ascertain

well-established histological features and to monitor tumor

dissemination histology was assessed from animals with large

tumors (>3 g) that were autopsied when they became

moribund. As shown in Fig 3A, most cells were large and

exhibited moderate-sized variation, round or oval nuclei, and

distinct nucleoli. Nuclear membranes were very prominent.

Mitotic activity was moderate. These cells appeared to be

interspersed with smaller cells with coarse nuclear chromatin

structure. Cytologically, two cell populations were teased

Fig 2. (A) Lung parenchyma shows micrometastases of largecells (hematoxylin and eosin stain; original magnification x 316;current magnification x 1 52). (B) Lymph node specimen showsdiffuse infiltration by large mononuclear cells that completelyeffaces the normal nodal architecture (hematoxylin and eosinstain; original magnification x240; current magnificationx 1 1 5). (C) Lung tissue section shows mononuclear cell infiltration

of the walls of a venule that is diffusing into the surrounding lungparenchyma. Note that the blood also contains large numbers ofthese cells (hematoxylin and eosin stain; original magnificationx 51 ; current magnification x 24).

XENOTRANSPLANTATION OF HCL 1515

Table 3. Lineage and Species Derivation of EH

and Molt-4 Tumors

Tumors

Antibodies EH Molt-4

Antihuman

T cells

OKT6 ND 46

LEU-1 1.5 45.5

B cells:

Bi 43.5 ND

Poly-V 40.5 0.5

HCL cells

CLL-1 47 ND

Antimouse:

T cells:

Thyl.2 16 20

H2

H-2Dk/H-2Kk/H-2Dd 38.5 33

Preirradiated (200 R/wk for 3 weeks) mice were implanted with EH

and MoIt-4 cells. When the tumors reached >2 g, cells were teased and

assayed by IFA with the antihuman antibodies OKT6, LEU-1, Bi,

anti-Igs. and CLL-1; and antimouse T cell (Thyl.2) and antimouse H2

complex anti-H-2Dk/H-2Kk/H-2Dd antibodies. Reactive cells are

expressed as a percentage of the 600 cells examined per assay.#{149}Poly..Vrefers to polyvalent antihuman Ig antibody.

H-2Dk/H-2Kk/H-2Dd-positive small cells (33%), more

than half of which also reacted with the MoAb Thyl .2.

Blood Counts

Fig 3. (A) Histological section of an SC HK tumor shows afairly homogenous population of large lymphoid cells with thicknuclear membranes and prominent nucleoli (hematoxylin andeosin stain; original magnification x 31 6; current magnificationx 1 52). (B) A cytocentrifuge spread of cells teased from the same

tumor shows moderate anisocytosis with a preponderance of

large. nucleolated lymphoid cells (May-Gr#{252}nwald-Giemsa stain;

original magnification x 400; current magnification x 192).

from primary tumors (Fig 3B): one, made oflarge immature

lymphoid cells; the other, reminiscent of small, mature

lymphocytes. Most but not all large cells exhibited tartrate

resistant acid phosphatase (TRAP) reactivity. Coimplanted

HT-1080 cells, recognizable histologically by their eosino-

philic cytoplasm and association with fibrous trabeculae,’8were absent in tumors. These nonproliferative cells appear

not to survive past 2 or 3 weeks postimplant.

Tumor immunophenotype. The species and lineage den-vation of cells teased from EH tumors was assessed by IFA

on two mice bearing tumors ranging in weight from 2 to 5 g.

Phenotypically, two cell populations were identified (Table

3): one of preponderantly large cells that reacted with

antihuman SIg (41%), B) (43%) and anti-cCLLa (47%)antibodies; the other made largely of small cells reactingwith antimouse MoAbs Thyl.2 (16%) and H-2Dk/H-2Kk/

H-2Dd (38%). Only background reactivity (2%) was

observed with the antihuman T cell antibody LEU-l. Like-

wise, primary tumors of two mice bearing Molt-4 tumors (2

to 4 g in weight) exhibited a preponderant population

(>45%) of OKT6- and LEU-l-positive large cells and

Complete counts from tail vein or heart puncture blood

were obtained in a group of ketamine-anesthetized nude

mice (n = I 2) before and three days after irradiation and 4

to 6 weeks after tumor implantation, respectively. TB!

induced 89% and 75% reductions in average circulating

lymphocyte (679/�zL v 4,096/�L) and platelet ( I 3 1 ,000/�L

V 478,000/j�L) counts, respectively (Table 4). Averages for

hemoglobin levels (15.6 v 14.0 g/zL) and granulocyte

(2,704/zL v l,777/jsL) and monocyte (135/zL v l05/�L)

counts were unaffected. Blood counts obtained at autopsy

(mean tumor size, 2.8 g) revealed a return to normal platelet

counts (mean, 584,000/�L), with little change in granulo-

cyte counts but the presence of anemia (mean, 1 2.Og/.zL)

and persistent lymphopenia (mean, 523/�L).

DISCUSSION

A preliminary experiment confirmed the widely held viewthat direct transplantation of human hematopoietic and

lymphoid malignant cells in nude mice is unsuccessful.

Naturally occurring cytotoxic antitumor antibodies25 and the

increased NK cell activity observed in nude mice23’24 havebeen implicated in the rejection ofsuch implants. In addition,

the tendency of leukemic cells to infiltrate local tissues rather

than form nodules may also impede implantation. However,xenotransplantation has been reportedly facilitated by host

preconditioning with TB!; coimplantation of nonprolifera-

tive, transplantation-promoting HT-l080 cells; or by both.’8’

21.29 Analysis of our cumulative data supports these findings.

Indeed, the overall frequency of “takes” increased from 8.3%

1516 FAGUET AND AGEE

Table 4. Effect of TBI and Tumor Implantation on Blood Counts

WBC PMN Lymph Mono Hb P)at

Pre-TBI 6,024 (1,729)

100

1,777 (94)

30(2)

4,096 (168)

68(3)

105 (15)

2(0)

14.0 (0.5) 478 (136)

Post-TBI 3,487(884)

100

2,704(84)

78(2)

697(49)

20(1)

135(44)

1(1)

15.6(0.7) 131 (19)

Postimplant 2,887 (493)

100

2,332 (77)

81 (3)

523 (73)

18 (3)

29 (13)

2 (0)

12.0 (1.1) 584 (154)

Tail vein or heart puncture blood was obtained from lightly ketamine-anesthetized mice and counted in a Coulter counter. WBCs. polymorphonuclear

cells (PMN). lymphocytes (Lymph), monocytes (Mono), and platelets (Plat, x 10�) are reported as means (SEM) of the absolute count per microliter and

as percentages (SEM) for each cell subset. Hemoglobin (Hb) is presented as mean g/dL (SEM).

‘These mice were studied sequentially post-TBI and postimplant.

in nonirradiated, non-coimplanted mice (n = 12) to 33.3%,

66.6%, and 97.4% in preirradiated mice receiving no coim-

plants (n = 12), unirradiated but coimplanted mice

(n = 12), and preirradiated coimplanted mice (n = 154),respectively, Chi-square analysis showed these differences to

be significant at P = .136, P = .0032, and P < .0001,

respectively. In addition, coimplantation in TBI-treated mice

was more effective than either preconditioning alone

(P = .0001). TB! proved moderately myelosuppnessive butprofoundly immunosuppressive as judged from platelet and

lymphocyte counts obtained postirradiation but preceding

transplantation, respectively. This observation supports the

view that TB! promotes tumor implantation’#{176} and metas-

tases.24 by reducing the vigorous NK cell activity reported in

nude mice.23’24 Similarly, the role of HT-1080 coimplantsmight be one of transplant facilitation through secretion of

growth, angiogenesis, or other promoting � Certaintumors inoculated IP reportedly trigger an earlier or more

potent activation of local or systemic host defense mecha-

nisms;’6 however, in our hands the route of inoculation

appeared to be less important for implantation because

tumors developed readily after IP or SC inoculation. Theshorter doubling time and cytogenetic abnormalities of HK27

seemed to confer no advantage over EH as judged by

implantation yield, tumor growth rates, frequency or distni-

bution of metastases, or host survival. This contrasts with

published data indicating that only aneuploid cell lines are

transplantable.TM Likewise, the newly established HK was astransplantable as the 3-year-old, multipassage EH, in con-

trast to published reports suggesting poor transplantability of

newly established cell lines.35

Our xenograft model exhibited a leukemic picture andheavy splenic infiltration reminiscent of human HCL;36’37

however, widespread micrometastases and macrometastaseswere more prevalent than the diffuse tissue infiltrationscharacteristically seen in the human and affected nonlym-

phoid organs. These metastases were evident throughout and

were easily demonstrable at autopsy. This contrasts with the

nonmetastatic nature of most heterotransplantation solidtumor models.’6’38 It might be significant that several hema-topoietic tumor models described to date also metastasize

spontaneously.21’22 This observation supports the view thatthe implantability and metastatic potential of tumors areinfluenced by a variety of host- and tumor-related factors’6

postulated nearly a century ago.3’ Cytotologically, cyto-

chemically, and immunophenotypically, primary tumors and

metastases were indistinguishable from the parent cell line

without apparent genomic dnift.�#{176}SC tumors were heavily

infiltrated by host cells as judged by their reactivity with

antimouse MoAbs H-2Dk/H-2Kk/H-2Dd. ‘This view is fur-

then supported by the observation that approximately 50% of

these infiltrative cells are of mouse T lineage (Thyl .2

reactive), which indicates an immune rather than a nonspe-

cific host reaction or a fibrosarcoma, a neoplasm rarely

associated with xenografts.4’ Dissemination of tumor cells

occurred mainly through hematogenous spread as seen on

tissue sections and as judged by immunophenotypic studies

of circulating mononuclear cells. Flow cytometry assessmentof the cellular DNA content of sequential retroorbital venousblood specimens confirmed hematogenous dissemination as

early as 1 week postimplant even in the absence of detectable

SC tumors (unpublished data). This supports the view thattumor dissemination does not necessarily arise from shedding

local tumors.22 This observation and the relatively large

number of tumor cells detected in blood further suggest that

at least a subset of these cells are not “in transit” but in fact

recirculate. By using such cell subsets, an animal model moreclosely resembling human HCL might be feasible; neverthe-

less, additional preconditioning or adaptation of the host or

cell lines or modification of inoculation procedures might be

required. Such a model would be most suitable for assessing

the potential usefulness of new therapeutic modalities for the

management of HCL; however, host-tumor histoincompati-

bility of xenotransplants and the frequent need for host

preconditioning to preclude early rejection suggest that such

models are not true representations of autochthonously

arisen tumors in humans in spite of apparent similarities.

Therefore, caution must be exercised when ascribing humanrelevance to data derived from such models. Nevertheless,

the usefulness of such models is evident, particularly forexploring novel experimental protocols that are not ethically

feasible in the human. In this context, our HCL animal

model has proved useful for evaluating the kinetics, target-

ing, and cytolytic activity (unpublished data) of MoAbs

developed against the cCLLa, an HCL-associated antigen.

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