(CANCER RESEARCH 50, 3078-3086. May 15, 1990]

Heterotransplantation of Human Lymphoid Neoplasms Using a Nude MouseIntraocular Xenograft Model1

Les White,2 Annette Trickett, Murray D. Norris, Vivienne Tobias, Leo Sostila, Glenn M. Marshall, and

Bernard W. StewartThe Children 's Leukaemia anil Cancer Research i 'nil /L. W., M. D. N., G. M. M., B. W. S.J and Departments of Haematology-Oncology ¡L.H'./, Anatomical Pathology[V. T.], and Electron-Microscopy ¡L.S.], The Prince of Wales Children's Hospital, Randwick, Sydney, New South Wales, 2031, and School of Paediatrics. ( niversity ofNew South Wales, P. O. Box 1, Kensington, Sydney, A'or South H ales, 2033 ¡L.W., B. H'. S.J, Australia

ABSTRACT

There are few effective models for the study of human lymphoidneoplasms, including in vivo xenografts in ¡mmunocompromisedanimals.Exploiting the additional immune privilege of the anterior chamber ofthe nude mouse eye, a novel method of direct heterotransplantation ofcells from childhood leukemias and lymphomas has been developed. Theestablishment and characterization of 18 lymphoid xenograft cell linesmaintained in the nude mouse intraocular model are reported. Cell sourcesfor heterotransplantation were specimens of bone marrow, peripheralblood, or lymphomatous masses obtained at either diagnosis or recurrenceof disease in the patients. The 18 patients and resultant cell lines weregrouped into four immunophenotypic categories: Category 1, B-lineage(pre-B and early pre-B), "common" acute lymphatic leukemias; Category

2, cell lines of similar immunophenotype derived from patients withunusual features; Category 3, B-cell neoplasms and cell lines; and Category 4, neoplasms and cell lines in part or totally of T-cell origin. Withreference to these groupings, rates of ingraftment from clinical specimensvaried according to immunophenotype and disease status: Category 1, 1of 15 at diagnosis, 5 of 7 at relapse; Category 2, 1 of 1 at diagnosis, 2 of2 at relapse; Category 3, 6 of 6 at diagnosis; and Category 4, 2 of 9 atdiagnosis, 1 of 1 with persistent disease. Rearrangements of the genesfor immunoglobulin heavy chain or Klight chain and for ,; subunit of theT-cell receptor gene were demonstrated according to immunophenotype,with the exception of one cell line which showed no rearrangements.Evidence of Epstein-Barr virus I)\A was shown in only one cell line, ofB-cell immunophenotype. Cytology, histopathology, and electron microscopy in representative patient and xenograft samples demonstrated correlations between the specimens of origin and cells or sections fromingrafted tumors in mice. It is concluded that the direct heterotransplantation of cells from childhood leukemias and lymphomas to the anteriorchamber of the nude mouse eye provides a relevant and reproduciblemodel for the maintenance and study of human lymphoid neoplasms.

INTRODUCTION

The heterotransplantation of human lymphoid neoplasms,particularly ALL,3 into nude mice is a potentially valuablecontribution to biologic research (1-7), in part due to thelimitations of in vitro methods (8-13). However, leukemiaxenografts have been persistently difficult to establish andmaintain utilizing methods effective for other malignancies (1,2, 4-6, 14-17). Additional immunosuppression of nude mice,most successfully achieved by a combination of splenectomyand total body irradiation, has improved ingraftment accordingto some reports (2, 5, 15, 16), although not in others (4).

Received 9/19/89; revised 1/22/90.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 U.S.C. Section 1734 solely to indicate this fact.

1This work has been funded in part by a research grant from the Prince HenryHospital Centenary Foundation. Sydney. Australia. Parts of these data have beenpresented at scientific meetings of the Clinical Oncological Society of Australiaand the International Society of Paediatric Oncology.

2To whom requests for reprints should be addressed.3The abbreviations used are: ALL, acute lymphocytic leukemia; NHL. non-

Hodgkin's lymphoma; AML, acute myeloid leukemia; CML. chronic myeloid

leukemia; MoAb, monoclonal antibody: FITC, fluorescein isothiocyanate; SAM,sheep anti-mouse immunoglobulin; EBV, Epstein-Barr virus.

Further manipulations, such as preculturing of leukemic cells(1, 16), addition of feeder cells (6, 17, 18), and intracranialinoculation utilizing the immune privilege ofthat site (19), havebeen reported, albeit with their distinct limitations.

Stemming from work in childhood retinoblastoma (20-23),White et al. (24) have developed a method for the heterotransplantation and maintenance of leukemic cells by direct inoculation into the anterior chambers of nude mouse eyes. We nowdescribe the utility and reproducibility of the model in 18additional newly established childhood lymphoid xenograftlines.

MATERIALS AND METHODS

Patient Material. Specimens from a total of 45 patients have beenprocessed in the laboratory for the purpose of establishing lymphoidcell lines. Eighteen patients diagnosed between December 1983 andJuly 1988 at ages ranging from 4 months to 14 years were the sourceof cells for successful xenografts. Clinical features, diagnosis, cytomor-phology, and immunophenotype, as well as disease status and type ofspecimen, are presented in Table 1. Eleven patients had ALL, four hadNHL, two were diagnosed as AML with some lymphoid expression,and one had a lymphoid crisis in CML.

Experimental Animals. Nude mice (nu/nu) of BALB/c background,were bred by the Australian Nuclear Science and Technology Organisation. They were maintained in a protected and closely monitoredenvironment in the laboratory (23, 25). All procedures were performedunder anesthesia and in accordance with permits from the Committeeon the Use of Animals in Research or Teaching of the University ofNew South Wales and guidelines issued by the National Health andMedical Research Council (Australia).

Heterotransplantation and Maintenance. Cells for heterotransplantation to nude mice were obtained from bone marrow, peripheral blood,or solid masses. For the former two, cells in Hanks' solution or sodium-

heparin were separated by the Ficoll-Hypaque method. The nucleatedcells were then washed and resuspended in RPMI 1640 at a concentration of 106-107 cells/ml. Solid masses were teased into cell suspension

by scalpels and pipeting and then washed and resuspended as above.The mice were anesthetized with pentobarbital (30 mg/kg i.p.) and

chloral hydrate (150 mg/kg i.p.). Four to six animals were used foreach clinical specimen or passage. Under a stereoscopic microscope(VM; Olympus, Tokyo, Japan) the eyes were held proptosed and 4 x103-8 x IO4 cells (total volume, 4-8 u\) suspended in RPMI wereinjected bilaterally into the anterior chambers through a 30-gaugeneedle (24, 25). The xenografts were subjected to microscopic examination on a weekly basis utilizing inhalational anesthesia (enflurane;Abbott Australasia Pty., Ltd.), and ingraftment as well as subsequentgrowth were monitored. Passage was carried out by enucleation oftumor-filled eyes prior to ulcération,teasing of cells in the mannerdescribed above, and resuspension in RPMI 1640. For the purpose offurther studies in vitro, or of passage, cells from one to six tumors ofeach harvest were pooled when necessary to provide adequate numbers.Passage s.c. was attempted by injection of 0.10-0.25 ml of suspensionthrough a 25-gauge needle.

Immunophenotyping. Peripheral blood and bone marrow sampleswere heparinized, diluted in Hanks' buffered salt solution, layered over

Ficoll-Hypaque gradients (density, 1.077), and centrifuged for 20 min

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XENOGRAFT MODEL FOR LYMPHOID NEOPLASMS

Table 1 Clinical origin of xenografted cell lines

PatientMMLMLLSM*TABERM'ABLFCSJLAMAABGWAMBGKLBAge(yr)7.11.313.83.51.813.13.70.43.110.45.39.86.66.811.88.413.35.4DiagnosisSexFMFMMMMFFMMMFMMMFMDiseaseALLALLALLCMLALLALLALLAMLALLALLALLNHLNHLNHLNHLAMLALLALLWBC*131,00077,80029.800NA8.400183.00010,90020.9008.4007.8008.1006,7009.0008,1006.30024,10014,800124,000StatusRelapseRelapseDiagnosisRelapseTesticular

relapseRelapseRelapseDiagnosisRelapseDiagnosisDiagnosisDiagnosisDiagnosisDiagnosisDiagnosisRelapseDiagnosisDiagnosisSourceMarrowMarrowMarrowMarrowBiopsyMarrowMarrowMarrowMarrowMarrowMarrowBiopsyBiopsyBiopsyBiopsyMarrowMarrowBloodCellsCytology'FABL2FAB

LIFABLIFABL2FAB

LIFABLIFAB

LIUndifferentiatedFAB

LIFABL3FAB

L3FABL3FAB

L3FABL3FAB

L3FAB

M3AFABLIFAB

LIImmunophenotypeC-ALL

(CM)C-ALLC-ALLC-ALL

(CM)C-ALLC-ALLC-ALL,

mixedfC-ALL/AML,mixedf~gNull-.

C-ALLfB-cellB-cellB-cellB-cellB-cellB-cellAML

->T-cell*T-cellT-cellCell

line*MMCL-4LMCL-5LLCL-15SMCL-16TACL-17BECL-18RMDL-13ABMCL-20LFCL-6CSBL-7JLBL-8AMBL-9AABL-10BGBL-11WABL-12MBMTL-3GKTL-14LBTL-19

" At diagnosis; NA. not available: C-ALL (CM),common ALL with intracytoplasmic immunoglobulin. pre-B phenotype. C-ALL, common ALL. early pre-B

phenotype.* Cell line nomenclature: patient initials (2 letters): abbreviated phenotype (3rd + 4th letter); L = leukemia or lymphoma; -laboratory number.' Cell type by French-American-British (FAB) classification.**Initial presentation CML; subsequent lymphoid crises (FAB L2) xenografted.' Patient with Down's syndrome; corresponding leukemic cells.^B-lineage variants of mixed or changing phenotype.'Two predominantly lymphoid cell lines from patients diagnosed as AML.

at 400 x g. Interface mononuclear cells were washed twice and resus-pended in Hanks' buffered salt solution containing 10% fetal calf serum.

Cell suspensions were prepared from solid tumor biopsies and nudemouse eyes by teasing and pipeting.

Immunophenotype was determined by fluorescence microscopy foreach of the 18 patients and all 18 xenografts (between passages 2 and6), using the MoAbs listed in Table 2. Mononuclear cells were labeledwith appropriately diluted MoAb by incubation for 20 min at 4°C.After washing, cells were stained with FITC-conjugated F(ab')2 SAM

(Australian Monoclonal Development, Artarmon, New South Wales,Australia) by a second 20-min incubation period. Cells were washed,mounted, and examined with an Olympus BH-2 fluorescent microscope. Control for mouse cell contamination involved use of irrelevantspecificity mouse antiserum followed by staining with SAM-FITC. Cellswith homogeneous FITC stain throughout the nucleus were identifiedas dead and excluded from analysis.

Table 2 Antibodies used far immunophenotyping

Antibodies12FMC8J5B4BlB2T6TilT3T4TlWM31T8NKH-1MY4MY7MY9SIgK\CD9101920211234578u

14wl333SpecificityH

LADRPre-B,plateletsPre-B,CALLA0Pan-BPan-BIntermediate

BCommonthymocytePan-T/E-rosettereceptorMatureTHelperTMatureTPan-TSuppressor

TNKPan

monocyteMyeloid,monocyteMyeloid,monocyteAnti-IgG,

-IgA,-IgM'lightchainsAlight chainsSourceCoulter"Flinders*CoulterCoulterCoulterCoulterCoulterCoulterCoulterCoulterCoulterWestmead

Hospital1'CoulterCoulterCoulterCoulterCoulterBehring'Dakopatts^Dakopatts

" Coulter Clone, Luton, Bedfordshire, England.* Flinders Medical Centre, Adelaide, South Australia.c CALLA, common acute lymphatic leukemia antigen; E-rosette. erythrocyte

rosette; NK, natural killer; SIg, surface immunoglobulin.•*Gift of Dr. K. Bradstock. Westmead Hospital. New South Wales, Australia.' Behringwerke AG. Marburg, West Germany.^Dakopatts A/S, Glostrup, Denmark.

Cytocentrifuged, methanol-fixed cell preparations were used for detection of intracellular markers. FITC-conjugated F(ab'>2 goat anti-IgM, (Cappel-Worthington, Malvern, PA) stained intracytoplasmicheavy chain of immunoglobulin during 20 min incubation. Presence ofterminal deoxynucleotidyl transferase was shown by 20 min incubationwith anti-human terminal deoxynucleotidyl transferase 1 (Supertechs,Bethesda, MD), 10 min wash, and 20 min incubation with FITC-SAM.

Cytological and Histológica! Analyses. Cells being processed forxenograft passage were resuspended in fetal calf serum and cytocentri-fuge slides were prepared (Shandon Scientific, London, United Kingdom). Cytomorphology and cytochemistry were analyzed utilizingstandard hematological methods with Wright's, periodic acid-Schiff,

Sudan black, esterase, and acid phosphatase stains. The immunocytol-ogy of individual cells was demonstrated by alkaline phosphatase anti-alkaline phosphatase method (26) with CD 10, CD 19, and CD22 (B-cell) as well as CD1 and CD2 (T-cell) MoAb (Dakopatts, Glostrup,Denmark).

Histopathological and electron microscopic analyses were performedon two representative specimens from each of the phenotypic groups.For light microscopy the enucleated eyes were fixed in buffered formalinfor 48-72 h and then bisected saggitally. The tissue was dehydrated ina series of graded alcohols, inbedded in paraffin, sectioned at 4 ^m,and stained with hematoxylin and eosin. Immunoperoxidase stainingwith MoAb (Dakopatts), to pan-B (L26, not clustered), pan-T (UCHL1,CD45R), and leukocyte common antigen (CD45) was performed onbuffered formalin-fixed paraffin sections cut onto poly-L-lysine slides,using the avidin-biotin complex technique (27). The antibody-bindingsite was located using diaminobenzidine as the chromogenic substanceand counterstained with Harris' hematoxylin. Appropriate positive and

negative controls were processed simultaneously.Electron microscopy was performed on cells which had been viably

frozen in RPMI 1640 and 10% dimethyl sulfoxide during passage.Cells were thawed, resuspended in glutaraldehyde, spun to a pellet(2000 rpm, 10 min), and fixed in Karnovsky's osmium ferrocyanide.

Preparations for analysis were made using uranium block and leadsection staining.

Molecular Analysis. High molecular weight DNA was extracted froma suspension of leukemic/lymphomatous cells harvested from the xenografts. Aliquots of 5 /ig were digested to completion with the appropriate endonuclease (EcoRl, BamHl), size fractionated over 0.8% aga-rose gels by electrophoresis and transferred to GeneScreen Plus membrane (New England Nuclear, Boston, MA) using an alkaline Southern

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XENOGRAFT MODEL FOR LYMPHOID NEOPLASMS

blot technique. The membranes were then hybridized to nick translated[32P]DNA probes of the immunoglobulin (heavy chain and Klight chain)or T-cell receptor (ßchain) genes. Human placenta! DNA was used ineach case as representative of germline configurations.

The heavy chain immunoglobulin gene (///) configuration was assessed using a 2.3-kilobase sau3A fragment of HuJH (28), while the «light chain gene (O) was investigated using a 2.5-kilobase EcoRl

fragment of the constant region of HuCk (29). Both of these probeswere obtained from P. Leder (Harvard Medical School, Boston, MA).The T-cell receptor ßchain gene (TCß)was analyzed using a 0.7-kilobase Pst\ fragment of a human complementary DNA (30), whichwas obtained from T. W. Mak (Ontario Cancer Institute, Toronto,Ontario, Canada). Finally, in order to detect whether EBV DNA waspresent in any of the xenografts, a probe containing an EcoRl EBVfragment cloned in plasmili pACYC184 was hybridized to the membranes (31).

RESULTS

Patient Material and Patterns of Ingraftment. Eighteen xen-ografted cell lines were established from a total of 45 specimensreceived in the laboratory (40% ingraftment). The rate of in-graftment varied according to both immunophenotype and disease status. Only 1 of 15 specimens from children with B-lineage "common" ALL (pre-B and early pre-B) taken at diag

nosis ingrafted, in contrast with 5 of 7 at relapse. Accordingly,of the six xenograft cell lines from patients with common ALLphenotype (Tables 1 and 3, first group) only one was derivedfrom bone marrow at diagnosis (LLCL-15), while the other fivewere from material obtained at relapse (three bone marrow,one peripheral blood, one testicular biopsy). Cytology was FAB-LI in four of the six patients and L2 in the other two (MM,SM), one of whom (SM) had ALL crisis on a background ofCML. The other five tended to also have, at initial diagnosis, adisproportionately high number of unfavorable prognostic features, with an average WBC of 86,000/mm3. All six expressedthe CD 10 antigen (Table 3): two also expressed intracyto-plasmic immunoglobulin (MM and SM, both L2 cytology) andmay be called pre-B, while the other four fit the early-pre-B

category.Three patients were the source of cell lines with phenotypic

manifestations of common ALL, in addition to other features(Tables 1 and 3, second group). One patient had Down's syn

drome and leukemia in relapse with LI cytology and B- as wellas some T-lineage expression (RM); one infant had undiffer-entiated AML at diagnosis expressing some lymphoid antigens(AB); and the third, with LI cytology at relapse, expressed noCD 10 and very little else (LF).

Six patients had either ALL (GS and JL) or NHL of B-cellimmunophenotype and FAB-L3 cytology (Tables 1 and 3, thirdgroup). Accordingly, tissues were obtained from solid tumor infour and bone marrow in two. All six cell lines were derived atdiagnosis from a total of six specimens (100% ingraftment) inpatients of this immunophenotype.

A total of ten specimens from leukemias with some T-cellexpression (nine at diagnosis, one with persistent disease) wereprocessed in the laboratory. Three patients were the source ofT-cell lines in the xenografts (Tables 1 and 3, fourth group).Two with conventional T-cell ALL, LI cytology, and someunfavorable prognostic features ingrafted in the mice at diagnosis. The third patient (MB) had presented with AML of FAB-M3a cytology and very minor T-cell expression. He showedrelative resistance to induction chemotherapy and a cell linecoexpressing some myeloid but mainly T-lymphoid antigenswas derived after two treatment courses (MBMTL-3).

Only six specimens from patients with AML were processed,inasmuch as the study primarily addressed lymphoid xenograft-ing. One of four attempts at diagnosis produced the cell lineexpressing myeloid, monocytoid, and lymphoid (B-lineage) antigens (ABMCL-20; Tables 1 and 3), and one of two fromrelapsed patients ingrafted producing a line with mixed T-celland myeloid expression (MBMTL-3; see above).

The rate of ingraftment per mouse eye injected with clinicalspecimens varied from 2 to 3 of 12 for common ALL to 9 to11 of 12 for B-cell neoplasms. In subsequent passage, the ratefor cell lines with poor initial ingraftment tended to increase(with the exception of SMCL-16, TACL-17, and BECL-18).

Growth of Xenografts. After a latency which varied from 3 to6 weeks, ingraftment was demonstrable by the presence of acloudy crescent at the weight-dependent extremity of the anterior chamber (Fig. 1). A grading system was developed, according to the proportion of the chamber occupied by the xenograft,which allowed sequential monitoring of growth (25). As thenumber of cells in suspension increased, the meniscus rose andthe cloudiness filled the chamber in 2 to 4 weeks (Fig. "LA).Eyesremoved at this stage contained 2-8 x IO6 cells. Subsequent

growth over 2 or 3 weeks caused distention of the eye (cellcount, 1-3 x IO7)and, if allowed to proceed, tended to ulcerate

and infiltrate the orbit as well as the central nervous system insome xenograft lines. Two xenograft lines (GKTL-14 andLBTL-19) metastasized to the cervical lymph nodes and alsowere the only ones to become passageable by s.c. injection afterthe initial intraocular ingraftment. All other s.c. passage as wellas all initial s.c. ingraftment failed.

The xenograft of B-cell lineage (RMDL-13; Tables 1 and 3)which was derived from a child with ALL and Down's syn

drome, displayed a radically different growth pattern. The cellsgrew in solid lumps throughout the anterior chamber, in themanner described with xenografts from solid tumors, particularly of neural origin (21-23). Three of the 18 xenograft lines(SMCL-16, TACL-17, BECL-18) were not able to be maintained beyond the third nude mouse passage (minimum, 6months).

Immunophenotype. The results of analyses by immunofiuo-rescence (quantitative and qualitative) of cells from both patientspecimens and intraocular xenografts are presented in Table 3.The 18 cell lines are in four groups: first group (6 cell lines)with common ALL (B-lineage) phenotype; second group (3 celllines) with similar phenotype but additional features; thirdgroup (6 cell lines) with B-cell phenotype; and fourth group (3

Fig. t. Nude mouse eye demonstrating suspension of ingrafted cells filling thelower quarter of the anterior chamber.

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XENOGRAFT MODEL FOR LYMPHOID NEOPLASMS

B •S«ir

Fig. 2. A, cross-sectional photomicrograph of the anterior chamber of the nude mouse eye (cornea above, lens below), demonstrating ingrafted lymphoid cellswithin the aqueous fluid. H & E. B, high power view of ingrafted human B cells with immunoperoxidase preparation using the leukocyte common antigen MoAb.The characteristic morphology as well as affinity of the MoAb for the cell membrane (dark coating) in a majority of cells are demonstrated.

cell lines) with T-cell phenotype. For each of the 18 cell linesthe immunophenotype of the patient cells is shown first, followed by that of the xenograft (Table 3).

Cytology and Histology. Cytomorphology and cytochemistryof xenografts in cytocentrifuge preparations showed lymphoidfeatures consistent with the cells of origin. Cell lines of commonALL and T-cell origin had relatively scant cytoplasm, occasional vacuoles and large nuclei showing open chromatin, oneto two nucleoli, and convolutions, seen particularly in the T-cells. The B-cell lines demonstrated abundant basophilic cytoplasm with multiple vacuoles and two to three nucleoli.

Light microscopy of histological sections from tumor-containing eyes demonstrated the presence and pattern of growthof lymphoid cells in the xenografts (Fig. 2). Growth was confined to the anterior chamber and lens capsule in early tumorswith invasion of ocular coats and posterior chamber in moreadvanced cases. Cells were fairly uniform with high mitoticrate. Xenografts of common ALL showed cell nuclei containingfine chromatin with small unconspicuous nucleoli and scantycytoplasm. The T-cells were similar except for more convolutednuclei. The B-cells showed coarsely clumped chromatin, one ormore prominent nucleoli, and pyrinophilic cytoplasm. Im-munostaining methods utilized in both the cytocentrifuge andhistological preparations demonstrated the presence of antigensin accordance with results obtained by immunofluorescence andpresented in Table 3 (Fig. 2B). Electron microscopy of individual cells confirmed features consistent with their malignant

human lymphoid derivation (Fig. 3).Molecular Analysis. The results of immunoglobulin and T-

cell receptor gene rearrangements are shown in Table 4 andFig. 4. The restriction digests utilized are indicated for eachprobe. Xenograft samples were assessed using the gene probesfor immunoglobulin JH and CKas well as the TC/i gene probe.All of the common ALL (B-lineage) and B-cell ALLs demonstrated JH gene rearrangements. Of the three T-cell ALLs, onlyxenograft MBMTL-3 displayed a rearranged JH gene followingBamHl digestion. CKgene rearrangements were evident in twoof the common ALLs (LMCL-5 and RMDL-13) and in four ofthe xenografts from B-cell neoplasms (CSBL-7, JLBL-8,AABL-10, and WABL-12). In addition, one patient from eachof these groups (MMCL-4 and AMBL-9, respectively) carrieddeletions of this gene. The presence of intact DNA was confirmed by reprobing with the JH gene.

The three T-cell ALLs displayed rearrangements of the TC0gene, whereas the other samples probed with TC/3 carriedgermline patterns (Table 4; Fig. 4). Only one xenograft(ABMCL-20) demonstrated a germline configuration for all ofthe gene probes. One xenograft (JLBL-8), from a patient withB-cell ALL, was shown to contain EBV DNA (Fig. 4).

DISCUSSION

In view of the recognized obstacles to xenografting childhoodlymphatic leukemias and lymphomas (1, 2, 4-6, 14-17), the

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XENOGRAFT MODEL FOR LYMPHOID NEOPLASMS

Fig. 3. Electron microscopy of representative cell (MMCL-4) from the common ALL (pre-B) immunophenotype group. The nuclear/cytplasmic ratio isrelatively high. Nucleus (jV)shows peripheral chromatin condensation, prominentnucleoli (n), and nuclear pocket (p) with ribosomes and endoplasmic reticulum.(The contents of the pocket are explained by postulating invagination of thecytoplasm in the nucleus.) The cytoplasm contains degenerate mitochondria (m),swollen vesicles of rough endoplasmic reticulum (ER), numerous ribosomes (R),and dense granules (arrows). Cell surface shows a dense projection (D). Karnov-sky's ferrocyanide, uranyl block, and lead section staining, x 16.000.

Table 4 Immunoglobulin and T-cell ßgene rearrangements

CelllineMMCL-4LMCL-5LLCL-15SMCL-16TACL-17BECL-18RMDL-13ABMCL-20LFCL-6CSBL-7JLBL-8AMBL-9AABL-10BGBL-11WABL-12MBMTL-3GKTL-14LBTL-19Immunophenotype

andoriginC-ALL"C-ALLC-ALLC-ALLC-ALLC-ALLC-ALL

(mixed)C-ALL(AML)C-ALL(Null)B-ALLB-ALLB-NHLB-NHLB-NHLB-NHLT-ALL

(AML)T-ALLT-ALLJHEcoRl/BamHlR/RR/GRR/GR/RR/DR/RG/GR/GRR/DR/RR/GRR/RG/RGGCV

ÄamHlDRGGGRGGRRDARGGGTCßEcoRl/BamHlGGGGGGR/AR/RR/R

" C-ALL, common ALL; G. germline; R. rearranged: A, allelic rearrangement;

D. deleted.

immune privilege of the anterior chamber has been utilized todevelop a reproducible model (24). The first established xeno-graft cell line (LALW-2) has been demonstrated to maintain itsmorphological (cytology, cytochemistry, histology), immuno-phenotypic, karyotypic, genotypic, electron microscopic, andingraftment characteristics for up to 14 serial passages (24, 25).Subsequent publications have addressed the molecular biologyof chromosome 11 pi3 translocation (32) and mechanisms ofdrug resistance (25, 33, 34) in the xenograft.

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Furthermore, a method for the evaluation of in vivo cytotox-icity has been evolved (25), based on experience with studies ofa variety of therapeutic agents and modalities in a comparablemodel for childhood retinoblastoma (20-23). A grading systemwhich, by direct visualization under a stereoscopic microscope,estimates the proportion of the anterior chamber filled byxenograft (Fig. 2A), has allowed serial evaluation of responseto chemotherapy administered to the mice (25). Given the lackof reproducible animal models for in vivo assays of cytotoxicityagainst human leukemias (3, 5-7), this application of the model

is also being further studied (25).The overall rate of ingraftment was 40% in this study. The

accuracy of this figure is limited in that not all patients diagnosed during the study were available as sources of specimens.Both the phenotype of the malignant cells and the status of thedisease in the patient influenced the probability of ingraftmentas well as subsequent maintenance. Most notably in the common ALL group, clinical evidence of drug resistance appearedto predict for increased ingraftment. At the opposite extreme,B-cell neoplasms never failed to ingraft and be maintained,regardless of disease status. Given the known difficulties inheterotransplantation of lymphoid neoplasms to nude mice (1,2, 4-6, 14-17), presumed to be primarily due to the limited buteffective residual immune competence of the host, the mechanisms whereby the above factors influence ingraftment have notbeen clarified. We are, however, considering methods for further improvement in xenografting efficiency.

Importantly, xenograft cell lines were demonstrated to beconsistent with the cells of origin and to maintain those characteristics after two to six mouse passages. However, somedifferences of immunophenotype were documented in the ingrafted cells, possibly due to selective pressures (Table 3). Bothpatients with common ALL and intracytoplasmic immunoglob-ulin (MM and SM) lost the latter in the xenograft, consistentwith a "left shift" to earlier in the B-lineage (early pre-B).

Similar but more subtle shifts are suspected in CSBL-7,ABMCL-20, and LFCL-6. In the six B-cell lines (Table 3, thirdgroup), the expression of surface immunoglobulin and specificlight chains differed somewhat from the cells of origin. Geno-typically, the B-cell lines were shown to have rearranged genesfor JH and CKin accordance with the above expression (Table4).

In some cell lines a small percentage of cells were fluorescentwith the mouse control antibody and a similar percentage falselyexpressed the inappropriate light chain (Table 3). The presenceof Fc receptors is considered to explain this non-specific result.Testing with monoclonal isotype controls indicated corresponding proportions of cells reactive with specific antibody subclasses. Therefore, MoAb of the appropriate isotype may bindto the corresponding Fc receptor. Because the sources of reagents for light chain detection and mouse control were poly-clonal (Table 2), many isotypes were represented in these reagents.

The derivation of a lymphoid (T-cell) line (MBMTL-3; Tables 1 and 3) from a patient with AML is of interest andconsistent with recent reports of leukemias of hybrid lineage(35-37). The model appeared to select for the lymphoid expression in the leukemic cells. Rearrangements of TCß,as well asJH were demonstrated in the MBMTL-3 xenograft (Table 4;Fig. 4). By contrast, the xenograft line ABMCL-20, the otherone derived from a patient with AML (Table 1), maintainedboth myeloid and lymphoid characteristics, while showing amarked increase in CD 10 expression compared to the cells oforigin (Table 3). Analysis of DNA did not reveal any JH, CKor

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XENOGRAFT MODEL FOR LYMPHOID NEOPLASMS

UH PROBE

C TA SM WA

UK PROBE

C RM MM CS AB

G-

G-

-R

Fig. 4. Representative Southern blot analyses using the immunoglobulin heavy chain(JH), a light chain (f »•).T-cell receptor rfchain(TTtf) and EBV probes. The JH. TCii, andEBV probed samples were digested withEcoRl, whilst the C«samples were digestedwith BamHl. Gcrmline patterns ((7) and rearranged (A'l bands for the immunoglobulin and

TCßgenes are indicated. The control (Q ineach case represents human placenta! DNA.Lanes representing xenograft samples are identified only by the patient initials. Samples RMand CS demonstrate rearrangements of C\,whereas sample MM carries a deletion. Onlyone of the 18 xcnografts (JL) displayed evidence of EBV DNA.

T-CELL PROBE

C GK LB LL CSEBV PROBE

C LM LF JL AM

G- •» «*-EBV

-R-R

G-

TCßrearrangements. It may be appropriate to redefine thisxenograft line as nonlymphoid with some possibly aberrantlymphoid expression. The biology of these apparently paradoxical leukemias is being further explored. The other xenograftsdemonstrated genotypes consistent with their immunopheno-typic lineage and monoclonality (Table 4; Fig. 4).

Two T-cell xenograft lines (GKTL-14 and LBTL-19) (Tables1 and 3) metastasized from the eye to the cervical lymph nodesand also adapted to s.c. passage after initial intraocular ingraft-ment. This pattern is the same as that reported with the LALW-2 (T-cell) xenograft in the original description of the model(24). In no instance was s.c. heterotransplantation successfulutilizing cells directly from the patient; this has been achievedonly by subsequent passage in the above three T-cell lines. Theimmune privilege of the anterior chamber appears to be ofparamount importance in the application of this model forxenografting human lymphoid neoplasms.

It is recognized that growth of cells from either Burkitt's

lymphoma, the equivalent FAB-L3 leukemia, or other B-cellneoplasms has been previously reported (18, 38-41). In vitroculture characteristics and limitations particularly with, but alsowithout, the influence of EBV (38-41), as well as requirementsfor xenografting (18) have been described. This is in contrastwith more persistent obstacles to successful xenografting ofother childhood lymphoid neoplasms of pre-B, early pre-B, orT-cell origin. Clinical behavior and presence of unfavorableprognostic factors have also been considered to be of relevanceto xenografting efficiency. One series reported no success withALL despite immunomodulation of the nude mice, while 4 of

18 patients with NHL ingrafted: all 4 patients died within 18months (4). Recent work using mice with SCID has producedingraftment (42) and immune function (43) of human maturelymphoid cells.

In vitro culture methods for lymphoid cells other than B-cellshave also suffered from limitations (8, 9) including low efficiency (8, 11); selection by growth factor specificity, phenotype.or karyotype (10, 11); and contamination by other clones (12,13). Establishment of long term growth of lymphoblastoid T-cells from patients with NHL and ALL has been achieved withthe use of human T-cell growth factor (44). Normal cells of T-phenotype have been transformed and maintained in culture bytransmission of human T-cell leukemia virus type II fromcocultured Mo-T cells (45). Continued refinements in methodology have produced a number of promising short term (13,46) as well as a longer term (47. 48) culture methods. However,given the intrinsic limitations, particularly growth factor requirements, of i/i vitro study, there remains an important placefor a reproducible xenograft model.

The intraocular xenografting method presented herein requires no immunomodulation of the nude mice. Xenografts canbe started from as few as 4 x 10' cells inoculated into the

anterior chamber. By contrast, s.c. implantation has been shownto require well in excess of IO6 cells and then ingraftment was

achieved only if the animals were both splenectomized andirradiated for each serial passage (5). Furthermore, our modelallows serial direct observation of tumor growth with potentialfor sequential evaluation of therapeutic responsiveness and longterm monitoring.

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XENOGRAFT MODEL FOR LVMPHOID NEOPLASMS

In order to consider the possible role of EBV transformationwith respect to successful xenografting, all of the 18 lines wereinvestigated for the presence of EBV DNA sequences. Only one(JLBL-8) showed evidence of EBV DNA. This finding in oneof the six patients with nonendemic B-cell neoplasia is consistent with established rates of association (49) and is not relevantto the efficiency of the model.

The reproducibility and potential benefits of the intraocularmethod for xenografting human lymphoid neoplasms have beendemonstrated. Further individual characterization, includingkaryotyping of the new cell lines, is being conducted. Forcollaborative projects it has been possible to transport cells,both at room temperature and frozen, to laboratories in Australia and overseas. One area of particular interest is the spectrum of cytotoxic agent responsiveness in the model and itscorrelation with prior exposure and clinical behavior in thepatient. Consequently, mechanisms of clinically relevant drugresistance have been and will continue to be investigated incurrent and future projects (25, 33, 34).

ACKNOWLEDGMENTS

The authors wish to acknowledge contributions to the manuscriptfrom Maria Kavallaris (photography) and Margaret Picone (typing).

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1990;50:3078-3086. Cancer Res   Les White, Annette Trickett, Murray D. Norris, et al.   Nude Mouse Intraocular Xenograft ModelHeterotransplantation of Human Lymphoid Neoplasms Using a

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