[CANCER RESEARCH 47, 3824-3829, July 15, 1987]

Rapid Growth of Human Cancer Cells in a Mouse Model with Fibrin Clot SubrenalCapsule Assay1

Howard J. Fingert,2 Zuying Chen, Neptune Mizrahi, Walter H. Gajewski,3 Michael P. Bamberg, and

Richard L. KradinDepartments of Surgery and Pathology, Massachusetts General Hospital Cancer Center, Boston, Massachusetts 02114

ABSTRACT

Rapid in vivo growth of cultured human cancer or leukemia cells wasachieved by implantation into the subrenal capsule of mice. A solidstructure, necessary for accurate implantation and measurement of tumorgrowth in this model, was provided by stepwise addition of fibrinogenand thrombin to the tumor cells, leading to rapid enzymatic formation ofa solid tumor-fibrin matrix. Human leukemia and epithelial cancersincreased in volume between 6- and 40-fold when measured 6-10 daysafter implantation into normal or immunosuppressed mice. Immuno-suppression of host CD-I mice was achieved by cyclosporine given dailyafter tumor implantation, cyclophosphamide given preimplantation combined with cyclosporine, or whole-body irradiation given preimplantation.Confirming the validity of tumor measurements, rumor histology in theimmunosuppressed mice revealed cell proliferation, invasion, and neovas-cularization. Similarly, no artifactual measurement of tumor growth wasobserved by nonviable cancer cells, implanted after in vitro exposure toa known cytotoxic concentration of thiotepa. This model provides aneconomical, short-term technique for the in vivo study of human tumorgrowth, for the evaluation of new cancer therapies, and for in vitro - invivo drug activity correlations in specific types of human cancer orleukemia cell lines.

INTRODUCTION

The examination of cultured tumor cells, derived from miceor other rodents, has provided insights into the biology andtreatment of cancer. Rodent cancers have been utilized widelybecause of their consistent growth capacity, both in cell cultureand in animal models. Unfortunately, the biological behaviorand therapeutic responses of commonly used rodent tumorsystems do not reflect those of common human malignancies(1).

Stimulated by the need for experimental models that moreclosely resemble human neoplasia, numerous cell lines havebeen established in serial passage from human cancer specimens. These cultured cells are used for investigations of newcancer therapies, based on the hypothesis that such results willcorrespond to the sensitivity or resistance of human cancersfrom which the tumor cells were derived (2). Although humantumor cell lines are useful for such in vitro experiments, theydo not generally provide practical models for experiments invivo. Preclinical development of cancer therapy requires theevaluation of drug combinations, pharmacokinetics, and thetherapeutic activities of metabolites that are generated in vivo(1). Furthermore, many new therapeutic strategies are targetedagainst biological interactions of cancer cells with the host (i.e.,tumor angiogenesis, invasion, and métastases),and these cannot be examined by using only cells in culture.

Congenitally athymic (nude) immunodeficient mice can beused as hosts for the propagation of human tumor cell lines,

Received 10/6/86; revised 3/17/87; accepted 4/15/87.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.

' Supported by USPHS Grants CA01157 and CA42802 and American Chemical Society Grant PDT-221C.

2To whom requests for reprints should be addressed, at Massachusetts GeneralHospital Cancer Center, Cox 6, Fruit St., Boston, MA 02114.

3 Lane Research Fellow in Gynecologic Oncology.

passaged as solid tumor grafts in the s.c. site. However, tumorgrowth in these animals often requires many weeks or months.Furthermore, many cultured tumor cell lines fail to producetumors in these mice, or do so with such low frequency as tomake this system impractical.

A short-term method for the growth of human tumors inmice was developed by Castro and Cass (3) and later refined byBogden et al. (4), using solid tumor fragments implanted underthe kidney capsule. SRC4 assay allows precise measurement

(accurate to 0.1 mm) of changes in tumor size, as judged by astereomicroscope with an ocular micrometer. A 7-fold greaterblood flow has been demonstrated for SRC-implanted as compared to s.c. tumors, suggesting that the SRC is advantageousfor delivery of nutrients and systemic antitumor agents (5).Some human tumors can be studied 4-6 days after implantationinto normal mice before immune rejection, providing economicadvantages over use of nude mice (4). Immunosuppression ofnormal mice further extends the duration of tumor growth andimproves the potential of the assays for discriminating activetreatments (6).

The SRCA has not been well defined with direct implantationof cultured cell lines or human leukemias, since this techniquerequires a solid matrix for accurate implantation and serialmeasurement. In this report, we describe a simple, rapid technique for direct implantation and quantitative analysis of cultured human cancer or leukemia cells in the SRCA. Our studiesdemonstrate that (a) stepwise additions of fibrinogen andthrombin provide a solid FC as a tumor cell matrix; (b) the FCmatrix maintains homogeneity and viability of implanted tumorcells; (c) rapid growth in vivo is observed by diverse types ofhuman epithelial or leukemia cell lines; and (</) these tumorsdisplay histológica! features that represent malignant growth invivo, including frequent mitoses, neovascularization, and invasion into normal tissues.

MATERIALS AND METHODS

Cell Cultures. A431 vulvar cancer (American Type Culture Collection), CX1 colon and LOX melanoma (NCI-Frederick Cancer ResearchCenter), and T24 bladder cancer (Dr. J. Fogh, Sloan Kettering Institutefor Cancer Research) were grown as monolayer cultures in RPMI 1640supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories), penicillin (100 units/ml), and streptomycin (100 ug/ml) at37*C in a humidified, 10% CO2 incubator. HL60 leukemia cells were

grown as a suspension culture in the same supplemented medium plusnonessential amino acids (Gibco) and L-pyruvate (Gibco), and thesecells were obtained after i.p. passage in athymic nude mice (A. Bogdenand W. Cobb, Bogden Laboratories). Human karyotypes of HL60 andT24 cells were confirmed in our laboratory, and the human karyotypeand non-HeLa isoenzyme pattern of other cell lines were establishedby the source laboratories. All cells were determined to be Mycoplasmafree by culture techniques.

Nonmalignant WI38 fetal lung fibroblasts (American Type CultureCollection) and FS2 foreskin fibroblasts (Dr. R. Sager, Dana-FarberCancer Institute; Ref. 7) were maintained in early-passage monolayerswith the same medium and culture conditions.

4The abbreviations used are: SRC, subrenal capsule; SRCA, subrenal capsuleassay; FC, fibrin clot; CSA, cyclosporine; CYT, cyclophosphamide.

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Fibrin Clot Formation. Fibrinogen (bovine; Sigma Chemical Co., Lot55F9305) was stored at -70'C in aliquots after dissolving in phosphate-

buffered saline (20 mg/ml). Optimal activity was achieved by slowdissolving over 1-2 h on a rotor, with care to avoid bubbling orclumping. Thrombin (bovine; Sigma, Lot 114 F-9461) was stored at-70°C in aliquots (20 units/ml) after dissolving in double-strengthDulbecco's modification of Eagle's medium (Gibco).

To provide a fibrin matrix, logarithmically proliferating cells werefirst obtained as a single cell suspension. Monolayer cultures weresuspended by treatment with trypsin-EDTA solution (Gibco) andwashed 3 times in complete medium to remove trypsin-EDTA andcellular debris. The cells were then counted and centrifuged at 100 x gfor 5 min to form a loosely packed cell pellet, and the supernatant wasdiscarded. To facilitate subsequent handling of the cell pellet andreagents, this last centrifugation was performed after transferring thedesired cell number with 1.2 ml complete medium into a 1.5-mlmicrocentrifuge tube (VWR Scientific). In a typical experiment, 1 xIO7centrifuged cells produced a pellet with a volume of about 250 pi;

15 ni fibrinogen (20 mg/ml) were added and dispersed throughout thecell pellet by gentle tapping; 8 pi thrombin (20 units/ml) were thenadded and dispersed in a similar manner. The added volumes did notexceed 10% of the total volume to ensure over 90% cellularity of theimplanted grafts.

The cell pellet was then incubated at 37"C for 5-10 min. The

thrombin produced enzymatic cleavage of the fibrinogen, resulting in asolid FC matrix surrounding the tumor cells. Using a sterilized, triangular metal spatula wetted with cold medium, the pellet was rimmedonto a Petri dish with RPMI 1640 supplemented with penicillin,streptomycin, and 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesul-fonic acid buffer (Sigma), then immediately cut with a no. 10 scalpelinto about 100 1- to 1.4-mm fragments for implantation. Thus, eachimplant contained about 1-2 x 10s cells, and the cell number per

implant could be monitored and modified by altering the number andsize of sectioned fragments.

SRC Assay. The SRC assay followed the technique described byBogden et al. (4). Female CD-I mice (24-28 g; Charles River Laboratories, virus and pathogen free) were anesthetized with chloral hydrate,placed on a sterile surface, and swabbed with ethanol-Zephiran chloridesolution. A 1 cm incision was made through the skin and body wall inthe region of the left kidney. The kidney was partially exteriorized anda small slit was made in the renal capsule. The implanted fragment wasloaded onto the tip of a 19-gauge trocar and inserted through the slitto deposit under the capsule. Immediately after implantation, thelongest and shortest diameters of the implant were measured in situwith a dissecting microscope, equipped with an ocular micrometercalibrated so that 10 ocular micrometer units equaled 1 mm. Thekidney was then replaced into the body cavity, and the incision wasclosed with sterile wound clips. The implantation procedure requiredless than 2 h for a typical experiment involving 30-40 mice.

Mice were randomly assigned to treatment groups (5-7 mice/group)after implantation. At the termination of assay, each animal wasweighed and sacrificed by cervical dislocation under anesthesia. Theabdominal cavity was exposed, and the left kidney was removed andplaced under the dissecting microscope for measurement of final tumorsize. Tumor sizes were expressed as the average of longest and shortestdiameters, as described by Bogden et al. (4), or as tumor volume by theformula (length x width x width x Vi).

Drugs and Irradiation. CSA was purchased from Sandoz Pharmaceuticals as a liquid suspension (100 mg/ml) and diluted in oil (Miglyol812; Dynimit Nobel) prior to injection. CSA injections (80-120 mg/kg of body weight) were done s.c. on a daily basis starting up to 2 daysafter tumor implantation. Cyclophosphamide (Cytoxan; Mead Johnson) was dissolved in saline immediately before use and injected i.p.(150 mg/kg of body weight) 24 h before tumor implantation. Whole-body irradiation was achieved with a General Electric Maxitron 250 X-ray at 60 rads/min, administered 4-16 h prior to implantation.

Histológica!Staining. Initial FC pellets and tumor-bearing kidneyswere fixed in buffered 10% formalin, embedded in paraffin, and serialsections were prepared and stained with hematoxylin and eosin. Inaddition, trypan blue studies were done with cells before, during, and

after the FC protocol and SRC implantation in order to assess grosschanges in cell membrane integrity and viability. These trypan bluestudies were also used to monitor the quality (initial cell viability) ofimplanted grafts between replicate experiments.

RESULTS

Histológica! and Gross Appearance of FC Cell Pellets. TheFC protocol was effective in providing a solid matrix for avariety of human cancer or normal cells, and no adverse changeswere observed in cell morphology (Fig. 1). After fibrin formation, the cell pellets were transferred rapidly into bufferedmedium and sectioned into 1-mm implants, and cell viabilitywas well preserved throughout the 1- to 2-h period needed forthe SRC implantation (less than 10% decrease in the numberof viable cells by trypan blue exclusion). As shown in Fig. 1,the cells maintained a discrete, solid structure under the kidneycapsule, and 3-dimensional growth was observed macroscopi-cally and quantified directly by changes in average diameter, orby the computed tumor volume.

To confirm the validity of tumor measurements, we investigated histológica! features in multiple cross-sections of formalin-fixed tissues. As shown in Fig. 2, these sections demon-

" -•• *. .7• •• » i ••l ' O «• . . I \

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BFig. 1. Histological and gross appearance of human tumor cells in the SRCA.

Cells were initially grown as monolayer or suspension cultures in RPMI 1640and 10% fetal calf serum. Logarithmically proliferating cultures were washed infresh medium, centrifuged to form a cell pellet, and fibrinogen and thrombin(bovine) were added as described in the text to produce a clot. The total addedvolumes did not exceed 10% of the cell volume to ensure over 90% cellularity ofimplanted grafts. A. cross-section of T24 human bladder cancer cells on day ofimplantation. Upper cells are tumor under the kidney capsule, lower cells arekidney. H & E, x 100. />',gross appearance of human tumors (HL60 leukemia

cells) under the kidney capsule on day 0 (left), day 6 (middle), and day 9 (right,angled view). Horizontal scale bar = 20 ocular micrometer units (2 mm). Three-dimensional growth was most evident after day 6 in CD-I mice immunosup-pressed with cyclosporine ( 120 mg/kg/day s.c. given days 2-8 postimplantation)and Cyclophosphamide (ISO mg/kg i.p. given 24 h preimplantation), or withwhole-body irradiation (650 rads given 4-16 h preimplantation).

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•*§

B

Growth of FC Implants versus Passaged Solid Tumors. Afterpassage in nude mice, human cancers commonly reveal markedheterogeneity in terms of viability and cellularity (9). Thisheterogeneity can lead to unreliable growth and passage intoother nude mice, posing a problem for studies that optimallyrequire uniform growth and passage. We studied implants fromthe CXI human colon tumor line, taken from monolayer cellculture and implanted by the FC technique, or from solid CX1tumor that grew after s.c. injection of the same cells intoathymic nude mice. Growth of the FC implants exceeded thes.c. passaged tumors, even on day 12 after implantation intoCSA-immunosuppressed mice (Fig. 3). Histológica! studies,performed on representative samples of s.c. passaged tumorgrafts (day 0 implants), revealed numerous areas of microscopicnecrosis, despite our preselection of "viable" tumor material by

gross appearance; the overall viability of the s.c. passaged graftswas also demonstrated by growth of new tumors after s.c.implantation of representative material into nude mice. Inaddition, the initial growth rates of A431, LOX, and HL60cells (Fig. 4) exceeded those in published reports with the sametumor cells or related sublines, implanted s.c. in nude mice (10-12). Taken together, these results suggest that the FC techniquecan improve the homogeneity of implanted tumor cells andsustain their growth capacity in vivo, especially when comparedto s.c. passaged tumors in nude mice.

Growth of Human Cancer or Leukemia Cell Lines. In normalmice, human cancer and leukemia cells of diverse histológica!types exhibited distinct growth rates on days 0-6 (Figs. 3 and4), followed by immune rejection. In contrast to this commonpattern of tumor regression on days 6-9 in normal mice, adramatic increase in tumor size was observed after day 6 inimmunosuppressed mice, demonstrated by growth in 3 dimensions (Fig. 1) and by the computation of tumor volume asshown in Fig. 4. Studies with A431 cancer cells demonstratedimproved growth on days 9-10 in mice immunosuppressedwith CYT plus CSA versus CSA alone (Fig. 4A). As shown inTable 1, no statistical difference was observed between tumorsizes in mice immunosuppressed by whole-body irradiation (650rads) versus CYT plus CSA (P > 0.2; Student's t test). Similar

results were obtained with HL60 cells in whole-body-irradiatedmice (data not shown).

In independent experiments, consistent growth was observedby the same cancer cell lines, implanted after multiple (1-10)

Fig. 2. Histology of human tumors in the SRCA. A, T24 tumor cells withmitotic figures (open arrows) at border with kidney on day 6. Tissues representedin I and /( were obtained from mice immunosuppressed with CYT alone 24 hpreimplantation. Closed arrows, capillary network. H & E, x 400. B, earlyneovascular changes in HL60 leukemia cells on day 6, demonstrated by thepresence of a capillary network (closed arrows) within the tumor. Open arrows,mitotic figures. H & E, X 400. C, by day 9, HL60 cells invade the kidneyparenchyma, demonstrated by tumor cells surrounding renal glomeruli (closedarrowheads) and tubules (open arrowheads). Tissues were obtained from micetreated with CYT plus CSA as described in Fig. 1. H & E, x 250.

strated several properties that characterize malignant growthin vivo, including frequent mitoses, neovascularization, andinvasion into normal tissues. Features of malignancy were mostclearly demonstrated in the immunosuppressed mice; histologies on days 5-6 in immunocompetent mice were difficult toevaluate due to the presence of mouse cells involved in graftrejection (6, 8).

30

20

10

06 12 O 6

Days Post ImplantFig. 3. Growth of CX-1 human colon tumor cells in the SRCA. Columns,

mean value of tumor sizes (average diameters); bars, SE (5-7 mice/group); 10ocular micrometer units = 1 mm. The broken line indicates the mean size on day0. A, solid CX-1 tumor obtained from nude mice demonstrated no significantgrowth in normal CD-I mice (hatched columns) or in mice treated with CSA. B,growth of CX-1 tumor cells obtained from cell culture and implanted into CD-Imice by the FC-SRC protocol.

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22

A431

Control

B

LOX

I

50

40

30

20

HL60

Days Post Implant Days Post Implant Days Post ImplantFig. 4. Growth of human tumor cells in the SRCA. Tumor volumes were computed by the formula (length x width x width x '¡)(31). Cell culture, FC and SRC

implantation techniques were done as described in text. Points, tumor volumes on various days after implantation relative to average initial volume on day 0; bars,SE. I. A431 vulvar cancer cells; B, LOX melanoma cells; C, HL60 leukemia cells. Tumor regression after day 6 in control (immuno-competent) mice correspondedto histological evidence for immune rejection (6, 8).

Table 1 Growth of human tumor or normal cells in the SRCAValues are the mean change in tumor size (average diameters, by ocular

micrometer units) ±SE, measured by change from base line (size on day 0). Cellswere passaged in culture up to 10 times between individual experiments. T24cells + thiotepa were exposed in culture to 40 fig/ml thiotepa for I h beforeimplantation, a treatment producing over 95% cell kill by clonogenic survival.Immunosuppression was achieved by CYT plus CSA for day 9-10 groups, asdescribed in Figs. 1 and 4, or by 650 rads whole-body irradiation administered4-16 h before tumor implantation (groups designated -t RT). NT, not tested; T24,bladder cancer; A431, vulvar cancer; FS2, foreskin fibroblasts; WI38, fetal lungfibroblasts.

Change in average tumordiameter (OMU)«

Experiment Cells Day 6 Days 9-10

Cancer123T24T24T24+21.4±2.0+24.7

±4.8+20.7±1.4NTNTNT

1234 <+RT)5 (+RT)

Normal123

1

T24 + thiotepa

A431A43IA431A431A431

FS2FS2FS2

WI38

-0.5 ±1.4"

+7.3 ±1.1+7.2 ±1.4+9.1 ±1.1

NTNT

-0.1 ±2.0+0.5 ±0.6

NT

+2.1 ±1.6

NT

+20.0 ±1.0+22.0 ±1.6+21.7 ±2.2+ 19.3 ±1.2+ 18.8 ±2.8

-4.7 ±0.4NT

-0.4 ±1.2

+2.3 ±1.6* OMU, ocular micrometer units.''I' 0.001 versus non-thiotepa-treated controls; Student's I test.

passages in cell culture (Table 1). However, large differenceswere observed in the final sizes of different cell lines (Fig. 4),which were unrelated to initial number or viability of cells inthe implants. Normal human fibroblasts did not show significant growth after FC implantation.

T24 cells treated before implantation with thiotepa (40 ng/ml for 1 h), a concentration that yielded over 95% cell deathmeasured by clonogenic survival in culture (13), demonstratedno change in tumor size above that measured at the time ofimplantation (Table 1). Coupled with the histological studies

(Figs. 1 and 2) that showed mitoses and other properties of invivo tumor proliferation, these results provide evidence that themeasured growth of FC implants represents the in vivo prolif-erative capacity of the tumor cells, in contrast to some artifac-tual changes in graft sizes.

DISCUSSION

In this report we describe a rapid, simple, and quantifiabletechnique for direct implantation and short-term growth ofhuman cancer or leukemia cells in mice. The ability of tumorcells to survive and grow from a fibrin matrix is consistent withother investigations in culture and in vivo. Dvorak et al. (14)described a "fibrin cocoon," formed from plasma proteins, that

envelops some tumor métastases,and suggested that it mayprovide a partial barrier to immune rejection. Fibrin has beenobserved in the earliest stages of many experimental tumormétastases(IS), although other studies are not consistent withthese (16). Several investigators have utilized tumor transplantation models that contain high-fibrin clots, formed from peritoneal or plasma constituents (17). Using the 6-day SRCA inimmunocompetent mice, Stratton et al. (18) reported consistentgrowth with malignant ascites implanted after the tumor cellshad been centrifuged and allowed to clot, with or without addedfibrinogen. Furthermore, some studies suggest that fibronectinand related fibrinopeptides, which are components of commercial fibrinogen preparations,5 can promote angiogenesis (19)

and tumor cell proliferation (15, 20).The SRCA has gained wide popularity as a short-term model

for evaluation of cancer therapy in vivo. Using highly cellularhuman cancers, obtained from s.c. passage in nude mice, reproducible growth can be achieved during the 6-day period aftertransplantation into immunocompetent mice (4,21). With suchpassaged tumors, several studies have demonstrated excellentcorrelations between the 6-day SRCA in normal mice andresults with the same tumors in the s.c. position of athymicnude mice (4, 11, 21). Major advantages of the 6-day SRCA

5R. L. Kradin, unpublished data.

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include (a) the economy of broad-scale experiments with normalmice versus athymic nude mice (in most laboratories, the costcan be over 10-fold lower than comparable studies with athymicnude mice), and (h) the rapid end point of 6 days, compared to30+ days typical for s.c. tumors in nude mice.

While the SRCA does provide several advantages with passaged human tumors, a major drawback for some laboratorieshas been the continued requirement for athymic nude micebearing s.c. passaged tumors, used as a source of human tumormaterial for initial implantation. The stringent requirementsand high expense to maintain even small numbers of athymicnude mice have limited such in vivo experimentation to the fewlaboratories or institutions that can support such specializedfacilities. In contrast, the FC-SRCA requires only standardfacilities for tissue culture and normal mice and, thus, shouldallow broader experimentation with human cancer or leukemiacells in vivo.

The dramatic improvement in tumor growth, observed in theimmunosuppressed mice on days 9-12, is consistent with otherstudies from our laboratory with solid human tumor grafts (6).Using monoclonal antibodies to various subsets of mouse lymphocytes, our immunoperoxidase studies suggested that CSA-sensitive T-cells (L3T4 positive) are a major component of graftrejection in the 6-day SRC model (6). However, the results withA431 cells (Fig. 44) indicate that CSA-treated CD-I mice retainsome degree of immunocompetence, which can be blockedfurther by CYT (22). CYT alone allowed tumor growth forabout 6 days, but graft rejection occurred by days 8-10 in micenot given CSA.

Our previous experiments also suggested that the longer (10-day) assay period could improve the capacity of the SRC assayto discriminate active treatments, since differences betweentreated versus controls are more readily assessed with the largercontrol tumors (6). Such differences also become important fordiscrimination of combination therapies versus single agents;our preliminary studies suggest that the evaluation of therapeutic synergism (13) is more readily achieved in the period ondays 9-12 after tumor implantation. Similarly, some investigators have reported definite histológica! evidence for drugresponse but minimal or no changes in tumor size after treatment with known active anticancer agents (8), and these studieshave led to some difficulty in the interpretation of what constitutes a positive therapeutic response, specifically in understanding how decreased tumor volume correlates with tumor celldeath (23). Our experiments with chemotherapy-pretreated tumor cells suggest a feasible approach to investigate such changesin tumor sizes on various days after treatment in vivo, relatedto cell death measured by parallel studies in culture.

The present histológica! studies also demonstrate that certainproperties of neoplastic growth in vivo (e.g., angiogenesis andinvasion) are more readily assessed during the period on days6-12 after implantation into the immunosuppressed mice.These results are consistent with the studies by Rybak et al.(24), who described consistent angiogenesis on day 11 afterimplantation of solid human tumors into the SRC of athymicnude mice. Using syngeneic or allogeneic rodent tumor grafts,Distelmans et al. (25) reported reproducible invasion on day 8,and proposed a scoring system to quantify effects of drugsagainst tumor invasion. Using total tumor growth and/or histológica! changes as therapeutic end points, the FC-SRCA inimmunosuppressed mice may provide a more economical technique for /// vivo evaluation of anticancer agents that are targeted against such biological properties of human tumor cells.

The problem of drug-drug interactions between some chem

otherapies and cyclosporine has been suggested by recent investigations (26, 27). While the mechanism for this interaction(and its relevance for various types of anticancer drugs) ispresently unclear, concurrent administration of both CSA andthe chemotherapy appears to be necessary for such effects. Toavoid such potential interactions, we have routinely administered cancer therapies on day 0 or 1 after tumor implantation,using short half-life agents which are effectively gone by thetime we start CSA on day 2. In some experiments, we utilizedbiological therapies (i.e., tumor necrosis factor) on the sameday with CSA, in order to study therapeutic response at a timeof early neovascularization; these latter studies demonstratedno increased toxicity to normal or tumor tissues compared tocontrols. Furthermore, our studies with whole-body irradiation,given before tumor implantation, support the studies by Basleret al. (28) that demonstrate effectiveness of this technique,providing short-term immunosuppression without drug-druginteractions. Other protocols for either short- or long-termimmunosuppression of mice have been described (2, 22, 29),and these may offer certain advantages under various experimental conditions.

Bennett et al. (26) reported significant toxicity by CSA alone,administered daily at doses of 60 mg/kg/day. In contrast, weroutinely used CSA in doses of 80-120 mg/kg/day with nosignificant toxicity compared to controls, measured by weightloss or abnormal renal histopathology. However, we utilized anoil-based solvent for CSA, a different weight and strain of mice,and a shorter period for CSA treatments (beginning after animals recovered from anesthesia and resumed water consumption), and several of these factors may account for the differences in toxicity. Preliminary studies with our oil-based CSAin 20- to 22-g BALB/c mice also revealed poor tolerance todoses above 60 mg/kg/day, suggesting that CSA regimens mustbe optimized for the weight and strain of mice used.

The advantage of in vivo models to develop new cancertreatments has been stressed by both clinical and laboratoryinvestigators (1, 3, 11, 23). While many studies utilize humancancer or leukemia cells in culture, the number of in vivo studiesthat evaluate the same therapies and human cancer cells arelimited by the expense of athymic nude mice, erratic growthand "take" rates, altered genetic properties of passaged tumors,

etc. The studies presented here suggest an economical, short-term technique to evaluate in vivo potency, metabolism, andoptimal combinations of new anticancer agents, in conjunctionwith investigations of their activity in specific human cancer orleukemia cells in culture. The CXI, LOX, and HL60 cell lines(Figs. 3 and 4) are currently utilized for similar preclinicalinvestigations by research institutions (11, 30, 31). Our positiveresults with A431 and HL60 cells also suggest uses for in vivostudies related to growth factors or differentiation, i.e., toextend experiments that commonly use these or related tumorcell lines in culture (10, 12). In addition, these studies mayprovide a practical experimental model to investigate host-tumor interactions, effects of the tumor cell microenvironment,and other biological properties of human cancer cells in vivo.

ACKNOWLEDGMENTS

We thank A. B. Pardee, A. E. Bogden, J. A. Stratton, and J. A.Bennett for helpful comments, and Patricia Donahoe and Don Kufefor independent and critical reviewsof histologies.

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1987;47:3824-3829. Cancer Res   Howard J. Fingert, Zuying Chen, Neptune Mizrahi, et al.   Fibrin Clot Subrenal Capsule AssayRapid Growth of Human Cancer Cells in a Mouse Model with

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