Comparison of Intrapulmonary, Percutaneous Intrathoracic ...otoxylin and eosin and subsequently evaluated for tumor or other histopathology. Animals expiring immediately from postoperative

(CANCER RESEARCH 48. 2880-2886, May 15, 1988]

Comparison of Intrapulmonary, Percutaneous Intrathoracic, and Subcutaneous

Models for the Propagation of Human Pulmonary and Nonpulmonary Cancer

Cell Lines in Athymic Nude MiceTheodore L. McLemore,1 Joseph C. Eggleston, Robert H. Shoemaker, Betty J. Abbott, Mark E. Bohlman,

Mark C. Liu, Donald L. Fine, Joseph G. Mayo, and Michael R. BoydDevelopmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute [T. L. M., R. H. S., B. J. A., J. G. M., M. R. B.J and Program Resources,Inc., {D. L. F.I, Frederick Cancer Research Facility, Frederick, Maryland; and Departments of Surgical Pathology [J. C. E.], Radiology [M. E. B.J, and Medicine[M. C. L.], Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT

The propagation efficiencies, growth patterns, histolÃ³gica! appearances, and roentgenographic demonstration of tumors derived from sixcontinuous human pulmonary tumor cell lines implanted intrathoracically(i.t.) and intrabronchially (i.b.) were compared with the conventional s.c.implantation method at three different tumor cell inocula (N = 184, i.b.;.V = 185, Â¡.t.;V = 180, S.C.). A tumor-related mortality of 100% was

noted when the six different human lung tumor cell lines, including A549adenocarcinoma, NCI-H125 adenosquamous carcinoma, NCI-H460 largecell undifferentiated carcinoma, \( '1-1169 small cell carcinoma, and NCI-

H358 and NCI-H322 bronchioloalveolar cell carcinomas, were implantedi.b. at a 1.0 x 10' tumor cell inoculum. A similar (92%) tumor-related

mortality was observed when these same lung tumor cell lines wereimplanted i.t. at a 1.0 x 10* tumor cell inoculum (P > 0.10), whereas

minimal (5%) tumor-related mortality was noted when cells from the sixdifferent cell lines were implanted s.c. (/' < 0.001). In addition, a dose-

dependent, tumor-related mortality was noted for either i.t. or i.b. implantation when lower (1.0 x 10* or 1.0 x 10*) tumor cell inocula were

employed. HistolÃ³gica! characteristics and growth patterns of tumorspropagated employing the three implantation techniques were closelycomparable for all three propagation methods and, in all instances,histolÃ³gica! appearances of the tumors were representative of the currenttumor cell lines from which they were derived. Approximately 30% ofthe lung tumors propagated i.t. grew in the chest wall and/or in the lungparenchyma as well as in the pleural space. In contrast, tumors propagated i.b. grew predominantly in the lung parenchyma. When five non-

pulmonary human tumor cell lines (including U251 glioblastoma, LOXamelamontic melanoma, HT-29 colon adenocarcinoma, OVCAR 3 ovarian adenocarcinoma, and adriamycin-resistant MCF-7 breast adenocarcinoma) were propagated i.b. or i.t., there was considerable site-specificvariability in tumor-related mortality depending on the tumor type. These

data demonstrate that both the i.b. and i.t. models should be useful forthe in vivo propagation and study of certain human pulmonary andnonpulmonary carcinomas as well as being advantageous for futurestudies of cancer biology and developmental therapeutics.

INTRODUCTION

Recently, there has been increased interest in the use of invivo models for the propagation of human tumors at organ-specific (orthotopic) sites in at In mie nude mice (1-6) andselected orthotopic models for the propagation of renal cellcarcinomas (1-4), certain brain tumors (6), colorectal carcinomas (1), and pancreatic carcinomas (5) in immunodeficientmice have been previously described. The conceptual validity ofthese models, as well as previously described metastatic tumormodels (1), was first suggested by the original "seed and soil"

hypothesis proposed by Paget in 1889 (7). According to this

Received 10/26/87; revised 2/10/88; accepted 2/17/88.The costs Â»Ipublication 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.

' To whom requests for reprints should be addressed, at Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute-Frederick Cancer Research Facility, Building 428, Room 63, Frederick, MD21701-1013.

theory, organ-site specific implantation of tumor cells is essential for optimal growth and progression of tumors in vivo.

A major impediment to the preclinical investigation of humanlung cancer, the number one cause of cancer-related adultdeaths and a major cause of morbidity and mortality in theU. S. (8-11), has been the absence of optimal in vivo animalmodels for this disease. Xenograft s.c. models (1, 12, 13) aswell as intrarenal implantation methods (2, 14-16) have previously been employed for the study of human lung carcinomas,but these models have not proven entirely satisfactory (1-3, 17,18). Orthotopic implantation of pulmonary cancers might provide an improved model for propagation and study of thesetumors. This concept is supported by a recent report by McLemore, et a/., which has described an i.b.2 model for propa

gation of human lung cancers in athymic nude mice (19). Thepresent report describes another approach to the propagationof human lung tumors whereby tumor cells are implanted i.t.in immunodeficient mice and compares this i.t. model withpreviously described i.b. and s.c. models.

MATERIALS AND METHODS

Human Tumor Cell Lines. The continuous human tumor cell linesemployed for these studies have been previously described with regardto their origin, characterization, and maintenance in a recent report byAlley et al., (20). Each cell line was propagated in vitro utilizing standardsterile culture techniques after recovery from cryopreserved seed stockprepared and maintained at the NCI-FCRF tumor repository (19).

Experimental Animals. Female athymic NCr nu/nu mice were obtained from the NCI-FCRF at approximately 4-6 weeks of age andwere free of known pathogens at the time of study. All proceduresdescribed below were performed in a pathogen-free barrier at NCI-FCRF.

i.b. Implantation. Animals were anesthetized in a Harvard smallanimal anesthesia chamber (Harvard Biosciences, Boston, MA) employing a 7% flow of nebulized metafane (Pitman Moore, Inc., Washington Crossing, NJ)/100% oxygen mixture. Implantation i.b. was thenperformed using either 1.0 x IO6, 1.0 x IO5,or 1.0 x 10" tumor cells,

in a 0.1-ml final volume of HBSS (NCI-FCRF Medium PreparationLaboratory) as previously described (19). Animals were subsequentlyreturned to holding cages and observed for tumor development.

i.t. Implantation Procedure. Animals were fully anesthetized as described above and i.t. injections were then performed at the lateraldorsal midaxillary line just below the inferior border of the scapulawith a 1.2-cm, 27-gauge needle. The needle was advanced approximately 5 mm through the chest wall into the pleura! space, and a tumorcell inoculum of 1.0 x IO6, 1.0 x 10*, or 1.0 x 10" cells was then

dispersed into the pleural space in a final volume of 0.1 ml HBSS (Fig.1). The procedure requires approximately 1 min for completion and isrelatively easy to perform. Prior to return to their holding cages, animalswere placed under heat lamps for approximately 10 min to maintainbody temperature.

2The abbreviations used are: i.b., intrabronchial; i.t., percutaneous intratho-racic; i.e., intracranial; HBSS, Hanks' balanced salt solution; NCI-FCRF, National Cancer Institute-Frederick Cancer Research Facility, Frederick, MD.

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on June 25, 2020. © 1988 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

COMPARISON OF INTRATHORACIC MODELS FOR LUNG CANCER

Visceral Pleura

Lung

B

Fig. 1. i.t. implantation. ,â€¢(,diagram of i.t. implantation of a tumor cellsuspension within the right pleural space. Note the location of the needle inrelationship to the visceral and parietal pleurae. B. demonstration of the localization of an i.t.-implanted radiopaque suspension within the pleural space bycontrast dye-enhanced radiography. The chest radiograph on the left representsan anterior-posterior view and the one on the right a right lateral view of a mouseimmediately following i.t. implantation with radiopaque dye. Note the localizationof the material to the right pleural space with delicate outlining of the pleurae bythe contrast material (solid arrows). A pneumothorax (open arrows) is also presentand is frequently observed following i.t. implantation. C, demonstration of thelocalization of a s.o. implanted radiopaque suspension in the s.c. space by contrastdye-enhanced radiography. The radiograph on the left is an anterior-posteriorview and the one on the right a right lateral view of a mouse following s.c.implantation with radiopaque dye. The contrast material is localized to the s.c.space (arrowy, shown best on the lateral view). Compare this with the i.t. method(Fig. IÃŸ)where contrast material is present predominantly in the pleural space.

S.C.Implantation Procedure. Inocula of 1.0 x IO7, 1.0 x IO6, 1.0 x10s, or 1.0 x 10" tumor cells in a 0.1 ml final volume of HBSS, were

placed s.c. in anesthetized mice in the lateral ventral midaxillary linewith a 27-gauge needle as previously described (19).

Chest Roentgenographic Studies. A Senographe SOOT mammographie radiological device (Thompson-CGR Medical Corporation,Columbia, MD) with a 0.1-mm focal spot with compatible screens,cassettes, and X-ray film (DuPont Lodose System; DuPont, Inc., Trenton, NJ) was employed for roentgenographic studies as previouslydescribed (19). GastrovistR radiopaque contrast material (Berlex Indus

tries, Wayne, NJ) was implanted i.t. or s.c. in a 0.1 ml volume forcontrast dye-enhanced radiographie studies (Fig. l, B and C). Animalswere immediately evaluated by anterior-posterior and/or right lateralchest roentgenography following injection of the contrast material.

Monitoring Animals for Tumor Development. Animals that receivedeither i.b., i.t., or s.c. implants were followed daily for signs of tumordevelopment. Those animals exhibiting tumor-related respiratory distress or debilitation from s.c. tumors were killed by CO2 inhalation andall animals not showing clinical signs of tumor within 200 days werekilled. The major organs (lung, liver, spleen, kidney, and brain) wereremoved and fixed in 10% buffered formalin and then processed forhistopathological evaluation. Paraffin sections were stained with hem-otoxylin and eosin and subsequently evaluated for tumor or otherhistopathology. Animals expiring immediately from postoperative complications and demonstrating no evidence of tumor represented approx

imately 5% of the animals studied for i.b. implantation and 8% for i.t.implantation; these were eliminated from the tumor-related mortalityevaluations and comparisons.

RESULTS

i.t. Implantation. Tumor-bearing animals became progressively cachexie and dyspneic following i.t. implantation andwith this technique, tumor cells first grew predominately in thepleural space and subsequently invaded the lung parenchymaland/or chest wall structures. An overall tumor-related mortalityof 92% was observed when the six lung cancer continuous celllines employed in the present studies were implanted i.t. at a1.0 x IO6 tumor cell inoculum (Fig. 2). Local mediastinal

invasion was observed with the i.t. model in approximately 20%of the tumor bearing animals. Occasionally distant mÃ©tastaseswere also observed for the i.t. implantation method (<1 %), withmÃ©tastasesoccurring in the left lung, and liver. Local invasionof the right chest wall structures was also noted in approximately 30% of animals following i.t. implantation. This wasprobably due to deposition of tumor cells in the puncture siteon withdrawal of the needle and/or to inadvertent puncture ofthe lung parenchyma during the i.t. procedure. HistolÃ³gica!appearances of tumors obtained from the i.t. implantationmodel were consistently similar to the parent tumor cell linesfrom which they were derived.

Comparison of i.b., i.t., and s.c. Models for Propagation ofHuman Lung Tumor Cell Lines. The i.b., i.t., and s.c. modelswere compared for efficiency of propagation of human lungtumor cell lines in a parallel study employing the six differentcontinuous human lung tumor cell lines, using three differenttumor cell inocula (W = 184, i.b., W = 185 i.t., N = 180 s.c.;Fig. 2). After a 1.0 x IO6 tumor cell inoculum, 100% tumor-

related mortality was observed for the i.b. technique and 92%for the i.t. implantation model, whereas minimal (5%) tumor-related mortality was observed when the s.c. tumor model wasemployed (P < 0.001, nonpaired one-tailed, Student's /-test).

The low s.c. mortality was most likely related to a lower s.c.tumor propagation efficiency since no surviving s.c.-implantedanimals had detectable tumors at the time of necropsy. Inaddition, when lower ( 1.0 x 10s or 1.0 x 104) tumor cell inoculawere employed, an inoculum size-dependent mortality was observed for both the i.b. and i.t. implantation methods (Fig. 2).

Comparison of the i.b. and i.t. implantation techniques generally demonstrated similar tumor-related mortality when a 1.0x IO6 tumor inoculum was employed (Fig. 2). There was,however, tumor-specific variation observed for the NCI-H358cell line, in that only 50% tumor-related mortality was observedwhen these tumor cells were implanted i.t. compared with 100%mortality for the i.b. implantation method at a 1.0 x 10'' tumorcell inoculum (Fig. 2). At 1.0 x 10s and 1.0 x 10" inocula, i.b.implantation generally yielded slightly steeper tumor-relatedmortality than the i.t. method. This was, however, also tumorcell line-dependent as demonstrated by the sharper mortalitycurve for the i.t. technique when cell lines such as the NCI-H460 and A-549 were employed (Fig. 2).

HistolÃ³gica! comparison of tumors obtained from the i.b.,i.t., and s.c. models was also performed (Figs. 3 and 4). In thecase of s.c.-propagated tumors for the NCI-H460 and NCI-H69 cell lines, a 1.0 x IO7tumor cell inoculum was required to

obtain s.c. tumors for comparison. These comparisons clearlydemonstrated that the i.t., i.b., and s.c. models yielded tumorswhich were characteristic of the original human lung cancercell lines, at least with regard to histolÃ³gica! appearance and

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NCI-H125

Fig. 2. Comparison of tumor-related mortality for athymic nude mice implanted i.b.,i.i.. or s.c. with various human lung carcinomacell lines. The figure depicts tumor-relatedmortality/unit time for i.b. (A), i.t. (â€¢)and s.c.(O) implantation of lung tumor cell lines at 1.0x 10' (A), 1.0 x 10s (A), or 1.0 x 10* (C)

tumor cell inocula. A minimum of 10 animalswere studied for each inoculum size and site.See text for statistical comparisons.

100

80

60

40

20

A549 NCI-H460

VA-1

20 40 60 80 100 120 140 160 180

Days Post Implant

NCI-H358

20 40 60 80 100 120 140 160 1Â»

Days Post Implant

NCI-H322

20 40 60 80 100 120 140 160 1Â»

Days Post Implant

NCI-H69

100

80

60

40

h

20 40 60 80 100 120 140 160 180

Days Post Implant

20 40 60 80 100 120 140 160 18

Days Post Implant

20 40 60 80 100 120 140 160 10

Days Post Implant

growth characteristics. As previously described (19), i.b. implanted tumors were localized to the lung parenchyma and werepredominantly located in the right lung. In contrast, i.i.-implanted tumors were frequently (in approximately 30% of theanimals tested) located in the right pleural space, lung parenchyma, as well as in the chest wall.

Roentgenographic techniques were also employed to evaluatehuman lung tumor growth following implantation using thethree different techniques. Radiological comparison of tumorspropagated i.b., i.t., and s.c. was performed using a low-dose,high-resolution mammographie radiological device (Figs. 5 and6). Right-sided lung tumors were easily demonstrated by roent-genography for either the i.b. or i.t. models (Fig. 5). Tumorspropagated s.c. were also easily distinguishable by X-ray analysis, but were not as distinct as those tumors arising in the thorax(Fig. 6). This technique was useful for identification of early

lesions in both the i.b. and i.t. models when right lateralroentgenographs were employed. Moreover, the progression ofthese tumors could be estimated radiographically over time(Fig. 5). The potential sensitivity of this method is furtherillustrated by its ability to define an infiltrate in the right lowerlobe of an animal immediately following i.b. implantation. ThisX-ray finding represents the anatomical location where thetumor cell suspension was i.b. implanted (see Fig. 5 for furtherdiscussion).

Comparison of i.b. and i.t. Models for Propagation of HumanNonpulmonary Tumor Cell Lines. Five human nonpulmonarycontinuous tumor cell lines were also evaluated for their abilityto be propagated i.b. or i.t. (Fig. 7). Interestingly, when theU251 cell line was implanted either i.b. or i.t. at a 1.0 x IO6tumor cell inoculum, 100% tumor-related mortality was notedand when this brain tumor was propagated orthotopically (i.e.),

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*.

V \% IÂ«

<* *

â€¢WVÂ»;^*,4.Â»v Â«r jL/a

V".â€¢'.mf'

I

ÃW ;

Fig. 3. Comparison of the histolÃ³gica]appearances of human tumor cell lineA549 propagated s.c. (A), i.t. (B), and i.b. (C). The tumor cells are cytologicallysimilar in the three locations, having abundant finely granular cytoplasm andnuclei with rather evenly dispersed chromatin. In addition, in all three locations,the tumor derived from a human adenocarcinoma demonstrates incomplete glandformation in its growth pattern. A, B, and C, 600 x.

Fig. 4. Comparison of the histological appearances of human tumor cell lineNCI-H358 propagated s.c. (A) i.t. (A) and i.b. (C). In all three locations, thetumor cells have pleomorphic nuclei and a moderate to large amount of cytoplasmwith, in some cells, prominent cytoplasmic vacuoles. Although derived from ahuman bronchioloalveolar cell carcinoma, the propagated tumor grows in solidsheets in all three locations. A, B, and C, 600 x.

90% tumor-related mortality was observed. With lower (1.0 X i.t. at a 1.0 x IO6 tumor cell inoculum, high (>70%) tumor-105 or 1.0 x IO4) tumor cell inocula, an inoculum-dependent related mortality was observed, whereas minimal mortality wasmortality was noted for all three implantation routes. Similarly, noted when these cell lines were implanted s.c. at a 1.0 x IO6when the LOX or the HT-29 cell lines were implanted i.b. or cell inoculum (Fig. 7; P < 0.001). Diminished mortality was

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Fig. 5. Roentgenographic appearance andprogression of the human lung carcinomaNCI-H460 cell line propagated i.b. or i.t. inathymic nude, mice. The radiograph progression of a tumor mass following i.b. implantation with a 1.0 x 10* tumor cell inoculum isdepicted from Day 1 (A) through Day 20 (/')employing right lateral X-ray views. An infiltrate (representing the localization of the tumor cell suspension) is present in the rightlower lobe on Day I following i.b. implantation(.I: arrows). The infiltrati- clears after 8 days(B) and a tumor mass appears by Day 14 (C;arrows). By Day 20, the tumor has progressedto a large mass involving the entire right lungand mediastinum (D, arrows). A large right-side tumor involving the mediastinum was documented at necropsy on Day 20. The rightlateral radiographie appearance of a tumormass following i.t. implantation is demonstrated in Fig. 5, E-H. The lung fields are clearon Day 1 following i.t. implantation (/â€¢.').A

small tumor mass is first seen on Day 9 (/;arrows) and progressively enlarges throughDay 18 at which time a large right-side lungmass is observed (G, H; arrows). A large right-side tumor involving the chest wall and pleuraewas noted at necropsy on Day 18.

Day 1

B

Day 8 Day 14 Day 20il Mi vimDay 1

B

Fig. 6. Roentgenographic demonstration of the human lung carcinoma NilH460 cell line propagated s.c. in an athymic nude mouse. Posterior-anterior (A)and right lateral (B) views of a tumor propagated via s.c. implantation with a 1.0x Id inoculum of the NCI-H460 large cell undifferentiated human lung carcinoma cell line. The tumor appears to be localized exclusively to the s.c. site,however, it is less well demarcated by the radiographie techniques when propagated s.c. compared with i.b. or i.t. implantation (Fig. 5).

also noted for i.b. or i.t. implantation of these lines at 1.0 xIO5or 1.0 x IO4tumor cell inocula. In contrast, minimal tumor-related mortality was noted when the MCF-7 ADR or theOVCAR 3 cell lines were propagated i.b. at 1.0 x IO6,or lower

tumor cell inocula (P > 0.30 in all instances). Mortality wasalso low when the ovarian carcinoma cell line was implantedi.t., but 80% tumor-related mortality was observed when thebreast carcinoma cell line was implanted i.t. at a 1.0 x K)'1

tumor cell inoculum.

DISCUSSION

The present report describes a different model for the in vivopropagation and study of human pulmonary cancers in iminu-

Day 9 Day 12

H

Day 18

1 11 i 1nodefÃ¬cientmice employing an i.t. implantation technique andcompares this model with the previously described i.b. model.Both the i.b. and i.t. tumor implantation procedures representthe first relatively short-term animal models for the efficient invivo propagation of human lung tumors. These techniques arerelatively easy to perform, are reproducible, and require a smallnumber of animals and minimal human lung tumor cells forexperimentation. These models also offer the advantage ofemploying human lung tumor cell suspensions rather thanrelying on less ideal carcinogen-induced animal tumors (21-27)or other nonhuman lung tumors (28). These models potentiallyallow investigators improved opportunity for study of thegrowth, progression, metastasis, and experimental therapeuticsof human lung tumors in vivo.

A potential disadvantage of the i.t. model is raised by theobservation that approximately 30% of all tumors propagatedby this method grow in the chest wall as well as in the pleuralspace and the lung parenchyma. This may limit the usefulnessof the i.t. model for selected in vivo drug testing studies andthose detailed tumor biology studies which require that thetumor cells grow in a localized intrapulmonar)- microenviron-

ment. In these instances, the i.b. model might provide a moreideal system for the in vivo investigation of human lung cancers(see Ref. 19 for discussion).

Greater tumor propagation efficiencies were observed for thei.b. and i.t. models compared with the previously described s.c.xenograft model, [which requires a >1.0 x IO7 tumor cell

inoculum for optimal tumor propagation efficiency (1, 12, 13,17)], when human lung tumor cell lines were implanted at low(1.0 x IO6) tumor cell inocula. The decreased tumor-related

mortalities associated with s.c. implantation in the presentstudies appeared to be directly related to the low s.c. tumorpropagation efficiencies observed for the tumor cell inoculaemployed. In addition, certain nonpulmonary tumors such asthe U-251 glioblastoma and the LOX melanoma cell lines werevery effectively propagated either i.b. or i.t. Interestingly, in thecase of the glioblastoma cell line, equally high propagationefficiencies were noted for both implantation of these tumorcells in the lung and for orthotopic (i.e.) implantation. Furthermore, previous studies have demonstrated that certain smallcell lung carcinoma cell lines are very effectively propagated i.e.at low (1.0 x IO6) tumor cell inocula (29, 30). These data,

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U251 LOX HT-29

Fig. 7. Comparison of tumor-related mortality in athymic nude mice implanted i.b., i.t.,i.e., or s.c. with various human nonpulmonarytumor cell lines. This figure depicts comparison of tumor-related mortality for five non-pulmonary tumor cell lines implanted i.b. (â€¢),i.t. (A), i.e. (Â»),or S.C(O) at 1.0 X 10' (A), 1.0X 10' (B), or 1.0 X IO4(C) tumor cell inocula.N = 10 for each implantation site at eachinoculum size. See text for statistical analyses.

r 20 40 60 80 100 120 140 160 180 200

Days Post Implant

20 40 60 80 100 120 140 160 180 200 20 40 60 80 100 120 140 160 180 200

Days Post Implant Days Post Implant

MCF-7 ADR OVCAR 3

rt -O 80

60

20 40 60 80 100 120 140 160 180 200

Days Post Implant

20 40 60 80 100 120 140 160 180 200

Days Post Implant

combined with the present observations, suggest that pulmonary and central nervous system tissues have similar microen-vironments which provide support for growth and progressionof certain lung as well as brain tumors. Conversely, othernonpulmonary tumors, such as the OVCAR 3, gave relativelylow propagation efficiencies when implanted i.b. or i.t.; furthersupporting the concept that an appropriate organ site-specificmicroenvironment might be essential for optimal tumor cellgrowth.

A differential propagation efficiency was also observed between the NCI-H358 lung tumor cell line implanted i.t. versusi.b. The tumor-related mortality was 50% after i.t. implantationcompared with 100% when tumor cells were implanted i.b. ata 1.0 x IO6 tumor cell inoculum. It is conceivable that the

Â¡ntrapulnionary site offers a selectively advantageous microenvironment for specific lung tumor cell types that is not providedwithin the pleural space. In this respect, it is also of interestthat certain nonpulmonary tumors, including the MCF-7 ADRbreast carcinoma cell line, grew more efficiently following i.t.rather than i.b. implantation at a similar tumor cell inoculum.

These data are consistent with the original seed and soilhypothesis proposed by Paget in 1889 (7) and suggest the

importance of organ site-specific (although not necessarily or-thotopic site-specific) tumor implantation for optimal tumorcell survival and growth. Positive and/or negative selectionfactors which are present in various mouse tissues appear to bepotentially important determinants of the success or failure ofhuman tumor cells to propagate in vivo (I, 31). These factorsare apparently important for the established human lung tumorcell lines, including both the small cell and nonsmall cellpulmonary carcinomas, as well as for certain nonpulmonarytumors. A number of tissue characteristics including the abundant blood supply in the lung, the relatively high compliance oflung tissue, and the availability of certain endogenous factorsin the lung which support more optimal tumor cell survival andgrowth (31-38), might all contribute to the enhanced survivaland growth of tumor cells in the immunodeficient mouse lung.

The present studies also describe the application of newnoninvasive X-ray procedures to periodically monitor lung tumor size for both i.b.- and i.t.-implanted tumors and these mayprovide an attractive approach for use in experimental therapeutics studies. Implantation of tumor cells into the right lungof nude mice is particularly advantageous since no other majoranatomical structures are located in this area which might

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interfere with radiographie evaluation of small tumors. Suchtechnology might ultimately provide a two-dimensional, radio-graphic approximation of tumor size which would allow for anoninvasive in vivo drug testing model which closely resemblesthe clinical setting in that it makes possible accurate, periodic,noninvasive monitoring of lung tumors following treatmentwith different therapeutic modalities. It also provides an experimental design whereby each animal serves at its own control.

In summary, this report compares two new models for the invivo propagation of human pulmonary and nonpulmonary tumor cell lines. These i.b. and i.t. models have specific advantagesover other conventional models and should be useful for futurei/i vivo studies of cancer biology and developmental therapeutics.

ACKNOWLEDGMENTS

The authors acknowledge the following individuals for providing celllines used in these studies: Drs. J. Minna and A. Gazdar (lung tumorlines), Dr. K. Cowan (breast carcinoma line), and Dr. T. Hamilton(Ovarian carcinoma line), NIH, Bethesda, MD; also Dr. O. Fodstad(melanoma cell line), Norsk Hydro's Institute, Oslo, Norway. We also

thank S. Jessee, J. Czarra, and M. Jones, for providing technicalsupport; K. Gill for the typing and organization of this text for publication; and Dr. J. Fidler, M. D. Anderson Hospital and Tumor Institute, Houston, TX, for his helpful advice regarding this manuscript.

REFERENCES

1. Fidler, I. J. Rationale and methods for the use of nude mice to study thebiology and therapy of human cancer metastasis. Cancer Metastasis. Rev., 5:29-49, 1986.

2. Fiebig, H. H., Weigeldt, H., Schuchhardt, E., Zeschigk, C., and Lohr, G. W.Transplantation of human tumors under the renal capsule of nude, immti-nocompetent, and preirradiated normal mice. In: H. D. Klein, H. KÃ¶ln(ed.),Adances in the Chemotherapy of Gastrointestinal Cancer, pp. 27-37. Erian-gen: perimed Fachbuch-Verlagsgesellschaft, 1984.

3. Naito, S., Von Eschenhach, A. C., and Fidler, I. J. Different growth patternsand biologic behavior of human renal cell carcinoma implanted into differentorgans of nude mice. J. Nati. Cancer lust., in press, 1988.

4. Naito, S., von Eschenhach, A. C., Giavazzi, R., and Fidler, I. J. Growth andmetastasis of tumor cells isolated from a human renal cell carcinoma implanted into different organs of nude mice. Cancer Res., 46: 4109-4115,1986.

5. Tan, M. H., and Chu, T. M. Characterization of the tumorigenic andmetastatic properties of a human pancreatic tumor cell line (ASPC-1) implanted orthotopically into nude mice. Tumor Biol., 6: 89-98, 1985.

6. Shapiro, W. R., Basler, G. A., Chernik, N. L., and Posner, J. B. Humanbrain tumor transplantation into nude mice. J. Nati. Cancer, 62: 447-453,1979.

7. Pagel, S. Secondary growths of cancer of breast. Lancet, /.- 571-573, 1889.8. Silverberg, E. Cancer Statistics, 1985. Ã‡Aâ€”ACancer Journal for Clinicians,

35: 19-35, 1985.9. Horm, J. W., AsirÃ©,A. J., Young, J. L., and Pollack, E. S. SEER Program:

Cancer Incidence and Mortality in the United States, 1973-1981. Bethesda,MD: Department of Health and Human Services, NIH Publication No. 85-1837, 1984.

10. Loeb, L. A., Ernster, V. L., Warner, K. E., Abbotts, J., and Laszlo, J.Smoking and lung cancer: an overview. Cancer Res. 44: 5940-5958, 1984.

11. American Cancer Society. 1986 Facts and Figures. New York: AmericanCancer Society, pp. 1-32, 1986.

12. Rygaard, J., and Poulsen, C. O. Heterotransplantation of a human malignanttumor to "nude" mice. Acta Pathol. Microbiol. Scand.. 77: 758-760, 1969.

13. Sharkey, F. E., and Fogh, J. Considerations in the use of nude mice forcancer research. Cancer Metastasis. Rev., 3: 341-360, 1984.

14. Aamdal, S., Fodstad, O., and Pihl, A. Human tumor xenografts transplantedunder the renal capsule of conventional mice. Growth rates and host immuneresponse. Int. J. Cancer, 34: 725-730, 1984.

15. Aamdal, S., Fodstad, O., and Pihl, A. Methodological aspects of the 6-day

subrenal capsule assay for measuring response of human tumors to anticanceragents. Anticancer Res., 5: 329-338, 1985.

16. Bogden, A. E., Haskell, P. M., LePage, D. J., Kelton, D. E., Cobb, W. R.,and Esber, H. J. Growth of human tumor xenografts implanted under therenal capsule of normal immunocompetent mice. Exp. Cell Biol., 47: 281-293, 1979.

17. Sharkey, F. E., Spicer, J. H., and Fogh, J. Changes in histological differentiation of human tumors transplanted to athymic nude mice: a morphometricstudy. Exp. Cell Biol., S3: 100-106, 1985.

18. Hoth, D., Marsoni, S., Rubinstein, L., Plowman, J., Wolpert, M., Simon,R., Leyland-Jones, B., and Wittes, R. The preclinical screen of the NCI drugdevelopment program: a correlation analysis with the clinical activity. CancerTreat. Rep., in press, 1988.

19. McLemore, T. L., Liu, M. C., Blacker, P. C., Gregg, M., Alley, M. C.,Abbott, B. J., Shoemaker, R. H., Bohlman, M. E., Litterst, C. L., I luhhurd.W. C., Brennan, R. H., McMahon, J. B., Fine, D. L., Eggleston, J. C, Mayo,J. G., and Boyd, M. R. Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice. Cancer Res., 47: 5132-5140, 1987.

20. Alley, M. C., Scudiere, D. A., Monks, A., Mursey, M. L., Czerwinski, M. J.,Fine, D. L., Abbott, B. J., Mayo, J. G., Shoemaker, R. H., and Boyd, M. R.Feasibility of Drug Screening with panels of human tumor cell lines using amicroculture tetrazolium assay. Cancer Res., 48: 589-601, 1988.

21. Blair, W. H. Chemical Induction of Lung Carcinomas in Rats. In: E. Karbe,J. F. Park (eds.), Experimental Lung Cancer, Berlin: Springer-Verlag, pp.199-206, 1974.

22. Delia Porta, G., Kolb, L., and Shubik, P. Induction of tracheobronchialcarcinomas in the Syrian golden hamster. Cancer Res., 18: 592-597, 1958.

23. Henry, C. J., and Kouri, R. E. Chronic inhalation studies in mice. II. Effectsof long term exposure to 2R1 cigarette smoke on C57BL/Cum x C3H/Anfeum) F mice. J. Nati. Cancer Inst., 77: 203-212, 1986.

24. Henry, C. J., Billups, L. H., Avery, M. D., Rude, T. H., Dansie, D. R.,Lopez, A., Sass, B., Whitmire, C. E., and Kouri, R. E. Lung cancer modelsystem using 3-Methylcholanthrene in inbred strains of mice. Cancer Res.,41: 5027-5032, 1981.

25. Ho, K., Wilcox, K., and FÃ¼rst,A. Pulmonary carcinogenesis by two arylhydrocarbons on three mouse strains. In: E. Karbe, J. F. Parks (eds.).Experimental Lung Cancer, pp. 62-71. Berlin: Springer-Verlag, 1974.

26. Paladugu, R. R., Shors, E. C., Cohen, H. H., Matsumura, K., and Benfield,J. R. Induction of lung cancers in preselected, localized sites in the dog. J.Nati. Cancer Inst., 65: 921-927, 1980.

27. Reznik-Schuller, H. M., and Gregg, M. Pathogenesis of lung tumors inducedby JV-nitrosoheptamethyleneimine in F344 rats. Virchows Arch. Pathol.Anat., 393: 33-341, 1981.

28. Staquet, M. J., Byar, D. P., Green, S. B., and Rozencweig, M. Clinicalpredictivity of transplant al>U:tumor systems in the selection of new drugs forsolid tumors: rationale for a three-stage strategy. Cancer Treat. Rep., 67:753-765, 1983.

29. Neuwelt, E. A., Frenkel, E. P., D'Agostino, A. N., Carney, D. N., Minna, J.

D., Harnet. P. A., and McCormick, C. I. Growth of human lung tumor inthe brain of nude rat as a model to evaluate antitumor agent delivery acrossthe blood-brain barrier. Cancer Res., 45: 2827-2833, 1985.

30. Chambers, W. F., Pettengill, O. S., and Sorenson, G. D. Intracranial growthof pulmonary small cell tumors in nude athymic mice. Exp. Cell Biol., 49:490-97, 1981.

31. Nicolson, G. Tumor cell instability, diversification, and progression to metastatic phenotype: from oncogene to oncofetal expression. Cancer Res., 47:1473-1487, 1987.

32. Erisman, M. D., Linnoila, R. L, Hernandez O., Diaugustine, R. P., andLazarus, L. H. Human lung small-cell carcinoma contains bombesin. Proc.Nati. Acad. Sci. USA, 79: 2379-2383, 1982.

33. Goustin, A. S., Leof, E. B., Shipley, G. D., and Moses, H. L. Growth factorsand cancer. Cancer Res., 46: 1015-1029, 1986.

34. Korman, L. Y., Carney, D. N., Citron, M. L., and Moody, T. W. Secretin/vasoactive intestinal peptide-stimulated secretion of bombesin/gastrin releasing peptide from human small cell carcinoma of the lung. Cancer Res., 46:1214-1218, 1986.

35. Moody, T. W., Pert, C. B., Gazdar, A. F., Carney, D. N., and Minna, J. D.High levels of intracellular bombesin characterize human small cell lungcarcinoma. Science (Wash. DC), 214: 1246-1248, 1981.

36. Roth, J., LeRoith, D., Shiloach, J., Rosenzweig, J. L., Lesniak, M. A., andHavrankova, J. The evolutionary origins of hormones, neurotransmitters,and other extracellular messengers. N. Engl. J. Med., 306: 523-527, 1981.

37. Sorensen, G. D., Pentengill, O. S., Brinek-Johnsen, T., Cate, C. C., andMaurer, L. H. Hormone production by cultures of small cell carcinoma ofthe lung. Cancer (Phila.), 47: 1289-1296, 1986.

38. Wood, S. M., Wood, J. R., Chatei, M. A., Lee, Y. C., O'Shaughnessey, D.,and Bloom, S. R. Bombesin, somatostatin and neurotensin-like immuno-reactivity in bronchial carcinoma. J. Clin. Endocrino!. Melali., 53: 1310-1312, 1981.

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1988;48:2880-2886. Cancer Res Theodore L. McLemore, Joseph C. Eggleston, Robert H. Shoemaker, et al. and Nonpulmonary Cancer Cell Lines in Athymic Nude Mice

PulmonarySubcutaneous Models for the Propagation of Human Comparison of Intrapulmonary, Percutaneous Intrathoracic, and

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