of 10/10
[CANCER RESEARCH 47, 5132-5140, October I, 1987) Novel Intrapulmonar}' Model for Orthotopic Propagation of Human Lung Cancers in Athymic Nude Mice Theodore L. McLemore,1 Mark C. Liu, Penny C. Blacker, Marybelle Gregg, Michael C. Alley, Betty J. Abbott, Robert H. Shoemaker, Mark E. Bohlman, Charles C. Litterst, Walter C. Hubbard, Robert H. Brennan, James B. McMahon, Donald L. Fine, Joseph C. Eggleston, Joseph G. Mayo, and Michael R. Boyd Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892 [T. L. M., M. G., B. J. A., R. H. S., C. C. L., W. C. H., R. H. B., J. B. M., J. G. M., M. R. B.]; Departments of Medicine, Radiology, and Pathology, Johns Hopkins University School of Medicine, Baltimore, Mailand 21205 [M. C. L., M. E. B., J. C. E.]; and Program Resources, Inc., National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701 IP. C. B., M. C. A., D. L. FJ ABSTRACT A major impediment to the study of human lung cancer pathophysiol- ogy, as well as to the discovery and development of new specific antitumor agents for the treatment of lung cancer, has been the lack of appropriate experimental animal models. This paper describes a new model for the propagation of human lung tumor cells in the bronchioloalveolar regions of the right lungs of athymic NCr-nu/nu mice via an intrabronchial (i.b.) implantation procedure. Over 1000 i.b. implantations have been per formed to date, each requiring 3 to 5 min for completion and having a surgery-related mortality of approximately 5%. The model was used successfully for the orthotopic propagation of four established human lung cancer cell lines including: an adenosquamous cell carcinoma (NCI- HI 25); an adenocarcinoma (A549); a large cell undifferentiated carci noma (NCI-H460), and a bronchioloalveolar cell carcinoma (NCI-H358). When each of the four cell lines was implanted i.b. using a 1.0 x 10*' tumor cell inoculum, 100 ±0% (SD) tumor-related mortality was ob served within 9 to 61 days. In contrast, when the conventional s.c. method for implantation was used at the same tumor cell inoculum, only minimal (2.5 ±5%) tumor-related mortality was observed within 140 days (P < 0.001). Similarly, when a 1.0 x IO5or 1.0 x Ml4cell inoculum was used, a dose-dependent, tumor-related mortality was observed when cells were implanted i.b.(56 ±24% or 25 ±17%) as compared with the s.c. method (5 ±5.7% or 0.0 ±0%) (P < 0.02 and P < 0.05, respectively). Most (>90%) of the lung tumors propagated by i.b. implantation were localized to the right lung fields as documented by necropsy and/or high-resolution chest roentgenography techniques which were developed for these studies. The intrapulmonar}- model was also used for establishment and propa gation of xenografts derived directly from enzymatically digested, fresh human lung tumor specimens obtained at the time of diagnostic thora- cotomy and representing all four major lung cancer cell types as well as a bronchioloalveolar cell carcinoma. Approximately 35% (10 of 29) of the fresh primary human lung tumor specimens and 66% (2 of 3) of tumors metastatic to the lung were successfully propagated i.b. at a 1.0 x lit1'tumor cell inoculum, whereas only 20% (1 of 5) of the specimens were successfully grown in vivo via the s.c. route from a 1.0 x 10" tumor cell inoculum. Our experience with this in vivo intrapulmonar} model for the orthotopic propagation of human lung tumor cells is consistent with the view that organ-specific in vivo implantation of human tumors facil itates optimal tumor growth. This new in vivo lung cancer model may substantially facilitate future studies of the biology and therapeutics of this catastrophic disease. INTRODUCTION Currently, lung cancer is the leading cause of cancer-related adult deaths in the United States, and its incidence continues to rise, especially in the adult female population (1-3). Ad vances in the study of the pathophysiology of human lung cancer as well as the discovery and development of new non- surgical treatment modalities for this disease may have been Received 1/5/87; revised 5/21/87; accepted 7/7/87. 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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed, at NCI-Frederick Cancer Research Facility, Bldg. 428, Rm. 63, Frederick, MD 21701. impeded by the lack of optimal animal models for use in these investigations (4). Animal models which use carcinogens to induce primary pulmonary tumors have been developed, but they have not been satisfactory for routine lung cancer studies (5-11). Major disadvantages of the carcinogen-induced animal models for study of primary pulmonary tumors include: they are time consuming (requiring a minimum of 6 mo for comple tion); and most importantly, they yield a variety of different histological tumor cell types which might not be directly rele vant to human lung cancer, and thereby limit their usefulness for meaningful biochemical, cell biology, and drug-testing stud ies (5-10). Athymic nude mouse models have recently been used for the in vivo propagation and study of human pulmonary cancers. The predominant prototypes which have been used for these studies are the s.c. xenograft (12-16) and the subrenal capsule (17-20) models. While these offer rapid and relatively simple methods for in vivo propagation of human lung tumors, they have limitations, particularly in their use for the study of lung cancer biology as well as for the discovery and development of effective treatment modalities for human pulmonary malignan cies (12, 15, 16, 20). A potential disadvantage of both the s.c. and subrenal capsule models for the propagation and study of human primary pul monary malignancies is that tumor cells are not orthotopically implanted (i.e., tumor cells are not implanted in the lung). Paget originally proposed that human tumor cell populations require organ site-specific interaction for optimal maintenance and progression (21). This concept has been widely supported by numerous studies using metastatic tumor models (for review, see Ref. 12) and by recent athymic nude mouse models which have been used to study the orthotopic propagation of selected human solid tumors, such as renal cell carcinoma (12, 16, 20, 22), pancreatic carcinoma (23), and certain brain tumors (24). A similar approach for the propagation of human lung neo plasms in the lungs of athymic nude mice would appear to offer a potentially very practical and relevant model for the study of this disease. This paper describes the successful development of a novel intrapulmonary model for the orthotopic propagation of human lung cancer cells in athymic nude mice using an i.b.2 implantation technique. MATERIALS AND METHODS Continuous Human Lung Tumor Cell Lines. Human tumor cell lines NCI-HI25, NCI-H358, and NCI-H460 were isolated from fresh solid 2 The abbreviations used are: i.b., intrabronchial; HBSS, Hanks' balanced salt solution; NCI-FCRF, National Cancer Institute-Frederick Cancer Research Fa cility, Frederick, MD; NCI-H460, NCI-H460 large cell undifferentiated human lung carcinoma cell line; NCI-H358, NCI-H358 human lung bronchioloalveolar human lung carcinoma cell line; NCI-HI25, NCI-HI25 adenosquamous cell human lung carcinoma cell line; A549, A549 human lung adenocarcinoma cell line; NCr-na/nw mice, NIH subline cancer research athymic nude mice homozy- gotic for the nude trait (this nomenclature registered with the Institute of Labo ratory Animal Resources, National Research Council, National Academy of Sciences, Washington, DC). 5132 Research. on February 26, 2020. © 1987 American Association for Cancer cancerres.aacrjournals.org Downloaded from

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  • [CANCER RESEARCH 47, 5132-5140, October I, 1987)

    Novel Intrapulmonar}' Model for Orthotopic Propagation of Human Lung Cancers

    in Athymic Nude MiceTheodore L. McLemore,1 Mark C. Liu, Penny C. Blacker, Marybelle Gregg, Michael C. Alley, Betty J. Abbott,

    Robert H. Shoemaker, Mark E. Bohlman, Charles C. Litterst, Walter C. Hubbard, Robert H. Brennan,James B. McMahon, Donald L. Fine, Joseph C. Eggleston, Joseph G. Mayo, and Michael R. BoydDevelopmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892 [T. L. M., M. G., B. J. A., R. H. S., C. C. L.,W. C. H., R. H. B., J. B. M., J. G. M., M. R. B.]; Departments of Medicine, Radiology, and Pathology, Johns Hopkins University School of Medicine, Baltimore,Mailand 21205 [M. C. L., M. E. B., J. C. E.]; and Program Resources, Inc., National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701IP. C. B., M. C. A., D. L. FJ

    ABSTRACT

    A major impediment to the study of human lung cancer pathophysiol-ogy, as well as to the discovery and development of new specific antitumoragents for the treatment of lung cancer, has been the lack of appropriateexperimental animal models. This paper describes a new model for thepropagation of human lung tumor cells in the bronchioloalveolar regionsof the right lungs of athymic NCr-nu/nu mice via an intrabronchial (i.b.)implantation procedure. Over 1000 i.b. implantations have been performed to date, each requiring 3 to 5 min for completion and having asurgery-related mortality of approximately 5%. The model was usedsuccessfully for the orthotopic propagation of four established humanlung cancer cell lines including: an adenosquamous cell carcinoma (NCI-HI 25); an adenocarcinoma (A549); a large cell undifferentiated carcinoma (NCI-H460), and a bronchioloalveolar cell carcinoma (NCI-H358).When each of the four cell lines was implanted i.b. using a 1.0 x 10*'tumor cell inoculum, 100 ±0% (SD) tumor-related mortality was observed within 9 to 61 days. In contrast, when the conventional s.c. methodfor implantation was used at the same tumor cell inoculum, only minimal(2.5 ±5%) tumor-related mortality was observed within 140 days (P <0.001). Similarly, when a 1.0 x IO5or 1.0 x Ml4cell inoculum was used,a dose-dependent, tumor-related mortality was observed when cells wereimplanted i.b. (56 ±24% or 25 ±17%) as compared with the s.c. method(5 ±5.7% or 0.0 ±0%) (P < 0.02 and P < 0.05, respectively). Most(>90%) of the lung tumors propagated by i.b. implantation were localizedto the right lung fields as documented by necropsy and/or high-resolutionchest roentgenography techniques which were developed for these studies.The intrapulmonar}- model was also used for establishment and propa

    gation of xenografts derived directly from enzymatically digested, freshhuman lung tumor specimens obtained at the time of diagnostic thora-cotomy and representing all four major lung cancer cell types as well asa bronchioloalveolar cell carcinoma. Approximately 35% (10 of 29) ofthe fresh primary human lung tumor specimens and 66% (2 of 3) oftumors metastatic to the lung were successfully propagated i.b. at a 1.0x lit1'tumor cell inoculum, whereas only 20% (1 of 5) of the specimenswere successfully grown in vivo via the s.c. route from a 1.0 x 10" tumor

    cell inoculum. Our experience with this in vivo intrapulmonar} model forthe orthotopic propagation of human lung tumor cells is consistent withthe view that organ-specific in vivo implantation of human tumors facilitates optimal tumor growth. This new in vivo lung cancer model maysubstantially facilitate future studies of the biology and therapeutics ofthis catastrophic disease.

    INTRODUCTION

    Currently, lung cancer is the leading cause of cancer-relatedadult deaths in the United States, and its incidence continuesto rise, especially in the adult female population (1-3). Advances in the study of the pathophysiology of human lungcancer as well as the discovery and development of new non-surgical treatment modalities for this disease may have been

    Received 1/5/87; revised 5/21/87; accepted 7/7/87.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.

    ' To whom requests for reprints should be addressed, at NCI-Frederick Cancer

    Research Facility, Bldg. 428, Rm. 63, Frederick, MD 21701.

    impeded by the lack of optimal animal models for use in theseinvestigations (4). Animal models which use carcinogens toinduce primary pulmonary tumors have been developed, butthey have not been satisfactory for routine lung cancer studies(5-11). Major disadvantages of the carcinogen-induced animalmodels for study of primary pulmonary tumors include: theyare time consuming (requiring a minimum of 6 mo for completion); and most importantly, they yield a variety of differenthistological tumor cell types which might not be directly relevant to human lung cancer, and thereby limit their usefulnessfor meaningful biochemical, cell biology, and drug-testing studies (5-10).

    Athymic nude mouse models have recently been used for thein vivo propagation and study of human pulmonary cancers.The predominant prototypes which have been used for thesestudies are the s.c. xenograft (12-16) and the subrenal capsule(17-20) models. While these offer rapid and relatively simplemethods for in vivo propagation of human lung tumors, theyhave limitations, particularly in their use for the study of lungcancer biology as well as for the discovery and development ofeffective treatment modalities for human pulmonary malignancies (12, 15, 16, 20).

    A potential disadvantage of both the s.c. and subrenal capsulemodels for the propagation and study of human primary pulmonary malignancies is that tumor cells are not orthotopicallyimplanted (i.e., tumor cells are not implanted in the lung).Paget originally proposed that human tumor cell populationsrequire organ site-specific interaction for optimal maintenanceand progression (21). This concept has been widely supportedby numerous studies using metastatic tumor models (for review,see Ref. 12) and by recent athymic nude mouse models whichhave been used to study the orthotopic propagation of selectedhuman solid tumors, such as renal cell carcinoma (12, 16, 20,22), pancreatic carcinoma (23), and certain brain tumors (24).A similar approach for the propagation of human lung neoplasms in the lungs of athymic nude mice would appear to offera potentially very practical and relevant model for the study ofthis disease. This paper describes the successful developmentof a novel intrapulmonary model for the orthotopic propagationof human lung cancer cells in athymic nude mice using an i.b.2

    implantation technique.

    MATERIALS AND METHODS

    Continuous Human Lung Tumor Cell Lines. Human tumor cell linesNCI-HI25, NCI-H358, and NCI-H460 were isolated from fresh solid

    2The abbreviations used are: i.b., intrabronchial; HBSS, Hanks' balanced saltsolution; NCI-FCRF, National Cancer Institute-Frederick Cancer Research Facility, Frederick, MD; NCI-H460, NCI-H460 large cell undifferentiated humanlung carcinoma cell line; NCI-H358, NCI-H358 human lung bronchioloalveolarhuman lung carcinoma cell line; NCI-HI25, NCI-HI25 adenosquamous cellhuman lung carcinoma cell line; A549, A549 human lung adenocarcinoma cellline; NCr-na/nw mice, NIH subline cancer research athymic nude mice homozy-gotic for the nude trait (this nomenclature registered with the Institute of Laboratory Animal Resources, National Research Council, National Academy ofSciences, Washington, DC).

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  • INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER

    Table 1 Histopathological characterization of surgically resected human lungtumor specimens propagated by i.b. or s.c. implantation

    CelltypePrimary*Squamous

    cell''AdenocarcinomaSmall

    cellLargecellLarge

    cell-smallcellBronchioloalveolarSubtotalMetastaticiAdenocarcinoma(prostate)Transitional

    cell (urinarybladder)SubtotalTotalNo.

    implanted

    i.b."12355132921332No.propa

    gatedi.b.*33111110

    (35)'112(66)12

    (38)No.

    implantedS.C."21101050005No.propa

    gatedS.C.*0000101(20)0001(20)

    " Fresh human tumor specimens were enzymatically digested and then implanted into athymic nude mice i.b. at a 1.0 x 10' tumor cell inoculum or s.c. ata 1.0 x 10' tumor cell inoculum as described in "Materials and Methods."

    *Tumors were classified as being successfully propagated only if they were

    sustained for at least 2 passages where passage 1 represents initial tumor implantation into nude mice.

    c Primary lung tumors.'' All diagnoses were histologically confirmed.' Numbers in parentheses, percentage of propagation efficiencies.' Tumors metastatic to the lung.

    Fig. 1. Diagram of the i.b. implantation technique. The shaded area on thediagram represents the caudal lobe of the right lung, the area where the majorityof tumor cells are localized following i.b. implantation.

    tumor specimens by the NCI-Navy Medical Oncology Branch usingstandard procedures (25), cultivated, characterized under a variety ofgrowth conditions (26), and kindly provided to the NCI-DevelopmentalTherapeutics Program by Dr. J. Minna and Dr. A. Gazdar. The A549cell line is a nude mouse ascites-passaged cell line derived at NCI-FCRF from the A549 cell line initially described by Giard et al. (27).

    The cell lines used for this study were propagated in vitro utilizingconventional sterile culture techniques after recovery from cryopre-served seed stock prepared and maintained at the NCI-FCRF tumorrepository. Tumor cells were thawed under sterile conditions, washed3 times with 0.9% saline, and counted with a hemocytometer; cellcounts were corrected for viability by the trypan blue dye exclusionmethod. Cells were cultivated in T75 cm2 flasks in conventional culture

    medium, consisting of RPMI 1640 (Quality Biologica Is. Gaithersburg,5133

    MD), 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT),and 2 mM L-glutamine at a density of 0.5 x IO6cells/10-ml/flask. Cellswere maintained at 37°Cin a humidified cabinet in an atmosphere of

    5% CÜ2in air. Cell monolayers approaching 75% confluency wereharvested using the DeLarco formulation of trypsin-EDTA and weresubcultured for a maximum of 8 to 12 serial passages. For i.b. or s.c.implantation experiments, cells were isolated by centrifugation at 500x g for 15 min, washed 3 times in 0.9% saline, suspended in a finalvolume of HBSS, and adjusted to appropriate inoculation densities. Allcell lines were documented to be of human origin by isoenzyme andkaryotype analyses and were verified to be free of Mycoplasma as wellas pathogenic mouse viruses at the time of study.

    Preparation of Fresh Human Lung Tumors. Fresh human lung tumorspecimens obtained at the time of diagnostic thoracotomy were mincedinto 0.5- to 1.0-mm3 pieces and enzymatically digested as previously

    described (28). Cell number, viability, as well as yield were then determined, and i.b. or s.c. implantations were performed as described below.All lung tumor diagnoses were histologically confirmed and are summarized in Table 1.

    Experimental Animals. Pathogen-free, female NCr-nu/nu mice orBALB/c x DBA/2 F, (hereafter called CD2Fi) immunocompetentmice, approximately 4 to 6 wk of age, were used. All proceduresinvolving the animals were performed in a pathogen-free barrier facilityat NCI-FCRF.

    Implantation i.b. Animals were anesthetized in a Harvard smallanimal anesthesia chamber (Harvard Biosciences, Boston, MA) attached to a standard Sonnifer anesthesiology apparatus (RichmondVeterinarian Supply Co., Richmond, VA) using a 7% flow of nebulizedMetafane (Pitman Moore, Inc., Washington Crossing, NJ)/100% oxygen mixture. A 1.0-cm ventral midline incision was made in the neckover the region of the trachea superior to the supraclavicular notch.The surgical procedures were facilitated by the use of a binocular x7 to10 magnification headset. The glandular tissue surrounding the tracheawas separated by blunt dissection, and the trachea was exposed bydissection of the peritracheal muscle sheath. The tissue between thetrachea! rings was then incised with the bevel of a 25-gauge needle, anda modified, 27-gauge, blunt-ended 1.2-cm needle was inserted into thetrachea and advanced into the right mainstem bronchus (Fig. 1). Aninoculum of 1.0 x IO6, 1.0 x 10s, or 1.0 x 10" tumor cells, in a 0.1-ml

    final volume of HBSS, was then introduced into the right mainstembronchus. Animals were maintained in a reverse Trendelenberg maneuver during the inoculation to ensure that the fluid containing the cellsremained in the distal airways of the right lung. The skin incision wasthen closed with surgical skin clips, and the animals were allowed torecover under heat lamps for approximately 10 min, were subsequentlyreturned to their holding cages, and were followed for clinical evidenceof pulmonary tumors.

    Implantation s.c. Tumor cells were implanted at a 1.0 x IO7, 1.0 xIO6, 1.0 x 10s, or 1.0 x 10* inoculum using the previously describeds.c. implantation techniques (12-14) in a 0.1-ml final volume of HBSS.

    Chest Roentgenographic Studies. A Senographe 500T 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 used for roentgenographic studies. (The 0.1-mm focalspot is essential for obtaining radiographs with high quality bone andsoft tissue detail). Gastrovist radiopaque contrast material (BerlexIndustries, Wayne, NJ) was used for contrast dye-enhanced radio-graphic studies to demonstrate localization of i.b.-implanted materialto the various lung fields. The standard i.b. implantation method(described above) was used for these studies with a 0.1-ml volume ofthe contrast material. Animals were then immediately evaluated byanterioposterior and right lateral chest roentgenography.

    Lung Tumor Cell Autoradiography Studies. Monolayer cultures ofNCI-H460 tumor cells were incubated with medium containing 10mCi/ml [3H]thymidine (specific activity, 18.2 Ci/mmol; New England

    Nuclear, Boston, MA) for 48 h prior to implantation. An inoculum of1.0 x IO6tumor cells was suspended in a 0.1-ml final volume of HBSSand then administered i.b. to NCr-nu/nu mice as described above.Control, radiolabeled NCI-H460 cells were incubated on glass cover-

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  • INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER

    Fig. 2. Demonstration of localization ofi.b.-implanted material to the right lung bycontrast dye-enhanced radiography. The chestradiograph on the left represents an anterio-posterior view, and the one on the right, a rightlateral view, of a mouse immediately followingi.b. implantation with radiopaque dye. Notethe localization of the contrast material to thecaudal (lower) lobe of the right lung (arrows).This corresponds to the highlighted area inFig. 1 representing the right lower lobe. Radiographs were performed as described in "Materials and Methods."

    -*- l'ß V

    Fig. 3. Localization of tumor cells to the bronchioloal-veolar regions of the caudal lobe of the right lung followingi.b. implantation. This light micrograph illustrates a paraffin-embedded, hematoxylin- and eosin-stained histologysection of the caudal lobe of the right lung of an athymicnude mouse l h post-i.b. implantation with 1.0 x 10" NCI-

    H460 tumor cells. A representative alveolus (a) and airway(o) are labeled for comparison. Note the clustering oftumor cells in the bronchioloalveolar regions with fillingof the alveolar spaces by the tumor cells. Representativetumor cells are demonstrated by arrows. Normal alveoliare prominent in the lower portion of the micrograph.X300.

    Fig. 4. Autoradiographic demonstration ofradiolabeled tumor cell distribution in the caudal lobe of the right lung. This light micrograph illustrates a periodic acid-Schiff-stained,hematoxylin-counterstained serial section ofthe caudal lobe of the right lung l h followingi.b. implantation with a 1.0 x IO6 NCI-H460

    tumor cell inoculum which was labeled for 48h prior to implantation with [3H]thymidine asdescribed in "Materials and Methods." Heav

    ily radiolabeled tumor cells are easily identifiedin the bronchioloalveolar regions (representative cells are demonstrated by arrows). Inset,control radiolabeled cells, placed on a glasscoverslip for I h. shown for comparison. Arepresentative alveolus (a) and airway (b) arealso labeled in the figure. Normal alveoli arepresent in the upper left and lower right portions of the micrograph.

    v •* fi*

    slips at 37°Cin the presence of 5% COj for l h and then processed for

    autoradiographic evaluation. Mice that received i.b. implants withradiolabeled cells were anesthetized with 0.1 ml of sodium pentobarbital(Richmond Veterinary Supply Company, Richmond, VA) l h after thei.b. implantation, and lung tissues were then fixed in situ by vascular

    perfusion with 10% low-molecular-weight dextran (Abbott Lab., Chi

    cago, IL) (w/v) in 0.9% saline followed by 2% glutaraldehyde in 0.1 Mcacodylate buffer (pH 7.4) as previously described (11). Individual lunglobes were collected and immersed in the above glutaraldehyde solutionfor 2 h, followed by further fixation in 10% buffered formalin for 2

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  • INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER

    "

    fl>T>^rV'^V "X-

    Fig. S. Light micrographs of microscopic NCI-H460 tumor nodules in the right lower lobe 7 and 10 days following i.b. implantation. These micrographsdemonstrate NCI-H460 tumor nodules (arrows) at 7 (A) and 10 (lì)days status posi-i.b. implantation. Note the location of the nodules in the peripheral regions ofthe right lower lobe. A, x 500; fi, x 200.

    days. Glass slides (Fisherbrand; Fisher Scientific, Fairlawn, NJ) wereprecleaned for 5 min with 70% ethyl alcohol containing 1% hydrochloric acid, rinsed well with running distilled water, placed in a 95% ethylalcohol rinse for S min, and subsequently air dried. Serial 5 ^ in sectionsof each lung lobe were placed onto precleaned glass slides at three levelsapproximately 100 urn apart. Duplicate sections were taken at eachlevel. The fixed lung tissues were subsequently embedded in paraffin,and autoradiography and staining procedures were performed as previously described (29). Lung tissue sections were then microscopicallyevaluated for radiolabeled tumor cell localization.

    Lung Tumor Cell Localization Studies. Athymic nude mice wereimplanted i.b. with 1.0 x IO6 NCI-H460 cells, and animals were then

    fixed in situ by vascular perfusion as described above at 1 h, 7 and 10days following implantation. Individual lung lobes were removed andseparated, embedded in paraffin, sectioned, stained with hematoxylinand eosin, and microscopically evaluated for tumor cell localization.

    Monitoring of Animals for Tumor Development. Animals that receivedeither i.b. or s.c. implants were carefully followed daily for signs oftumor development. Throughout the observation period, animals developing respiratory distress were killed by cervical dislocation. Allthose animals remaining after 140 days were also killed, and the majororgans (lung, liver, spleen, kidney, brain) were removed, fixed in 10%buffered formalin, and then processed for histopathological evaluation.Paraffin sections were stained with hematoxylin and eosin and thenevaluated for the presence of tumor or other histopathology. Mortalitydata were based on only those animals that died from direct effects oftumor growth and/or metastatic disease before 140 days.

    RESULTS

    The i.b. implantation method (Fig. 1) provides for the inoculation of human lung tumor cell suspensions into the right

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  • INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER

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    Days Post ImplantFig. 6. Comparison of tumor-related mortality from i.b. versus s.c. implanta

    tion of 4 human lung tumor cell lines in NCr-nu/nu mice. The results of mortalityfor i.b. and s.c. implantations are summarized in Fig. 6, where A to D representmortality from NCI-HI25, A549, NCI-H460, and NC1-H358 cell lines, respectively. »,1.0 X IO6;A, 1.0 x 10s; andO, 1.0 x IO4tumor cell inocula. Each pointrepresents animal mortality/unit time. Statistical comparisons of the i.b. (n =251) and s.c. (n - 120) implantation routes for the 4 human lung tumor cell linesat the different tumor cell inocula are as follows: 1.0 x 10»,P< 0.001 (nonpaired,one-tailed Student t test); 1.0 x 10', P < 0.02; and 1.0 x IO4, /»< 0.05. A, n =20, «= 21, and«= 30 for 1.0 x IO4, 1.0 x 10', and 1.0 x 10*tumor cell inocula,respectively; B, n = 10, n = 30, and n = 20 for 1.0 x 10", 1.0 x 10*, and 1.0 xIO4tumor cell i.b. inocula, respectively. C and D, n = 20 for all three i.b. tumorcell inocula; n = 10 for the 3 different s.c. tumor cell inocula for the 4 differentcell lines tested. The low mortality for s.c. implantation was related to the lowtumor propagation efficiencies.

    mainstem bronchus of athymic nude mice. Over 1000 i.b.implants have been performed to date, each requiring an averageof 3 to 5 min for completion and having a surgery-relatedmortality of approximately 5%. This model provides an ortho-

    topic site for tumor implantation in that the majority of tumorcells are deposited in the bronchioloalveolar regions of the rightlung, as verified by radiopaque dye-enhanced radiographie studies, routine histology, and autoradiographic evaluation (Figs. 2to 4).

    Radiopaque dye-enhanced radiographie studies were initially

    performed, whereby radiopaque contrast material was implanted i.b., and low-dose, high-resolution chest radiographs

    were then immediately performed. These studies clearly demonstrated radiographie localization of the radiopaque contrastmaterial predominantly to the caudal (lower) lobe of the rightlung (Fig. 2). Similarly, microscopic evaluation of histological

    sections of individual mouse lung lobes following i.b. implantation of 1.0 x IO6 NCI-H460 cells demonstrated localization

    of the majority of the tumor cells to the bronchioloalveolarregions of the caudal lobe of the right lung (Fig. 3). Localizationof tumor cells to this region of the right lung l h after i.b.implantation was more clearly demonstrated when 1.0 x IO6radiolabeled NCI-H460 cells were used (Fig. 4). Again, onlythe caudal lobe of the right lung consistently contained radio-labeled tumor cells when individual lung lobes were separatelyevaluated. In addition, microscopic tumor nodules were observed in predominantly the right lower lobe 7 and 10 daysfollowing i.b. implantation when individual lung lobes wereagain evaluated at these longer time intervals (Fig. 5). Thesedifferent studies all clearly demonstrated that the majority oftumor cells initially resided in the bronchioloalveolar regionsof the caudal lobe of the right lung following i.b. implantation.

    Athymic nude mice implanted i.b. with human lung tumorcells demonstrated clinical symptoms of cachexia and dyspneaas the pulmonary xenografts became progressively larger. Thesewere similar to the symptoms frequently observed in humanlung cancer patients. The tumor cells grew most frequently inthe peripheral regions of the right lung following i.b. implantation (Fig. 5); however, certain tumors did grow predominatelyin the airways (Fig. 9). Implantation i.b. produced tumors thatultimately led to the death of the animals (Fig. 6). Lung tumorprogression could also be readily followed roentgenographically(Fig. 7), thus allowing tumor-bearing animals to be monitorednoninvasively. When the 251 tumor-bearing animals represented in Fig. 6 were studied at necropsy following i.b. implantation, 91% of the tumors implanted by this method werelocalized to the right lung. Other tumor locations included theleft lung (1%), trachea (2%), or peritracheal (6%) area. Localmediastinal invasion was observed at necropsy in approximately90% of the tumor-bearing animals following i.b. inoculation.In 3% of the i.b.-implanted, tumor-bearing animals, distantmétastaseswere also observed at necropsy in the left lung,lymph nodes, liver, or spleen.

    The histologies of tumors obtained from the i.b. implantationmodel were consistently similar to the parent human lung tumorcell lines from the which they were derived (n = 251). As anexample, a tumor obtained from the right lung of a nude mouse19 days following i.b. implantation with the NCI-H460 cell linedemonstrated characteristic histopathological features of a human large cell undifferentiated carcinoma of the lung (Fig. 8).

    The reproducibility of this model among different cell lineswas demonstrated in that it produced similar pulmonary tumorsand death patterns for the 4 lung tumor cell lines tested (Fig.6). Reproducibility within cell lines was also demonstrated forthe NCI-H460 cell line when it was implanted i.b. multipletimes over a 6-mo interval (coefficient of variation for meansurvival was 24% for this cell line implanted on 4 separateoccasions).

    The intrapulmonar) model was also compared to the conventional s.c. model for effectiveness in propagation of human lungtumor cells. When the 4 different human lung tumor cell lineswere implanted i.b. at 3 different tumor cell inocula, 100%tumor-related mortality was observed at the 1.0 x IO6 tumorcell inoculum, and a dose-dependent response was noted withlower (1.0 x 10s or 1.0 x 10") tumor cell suspensions (Fig. 6).In contrast, minimal (5%) tumor-related mortality was observedwithin 140 days for the s.c. model for the 4 cell lines at alltumor cell inocula tested (Fig. 6). When tumor cells from the 4cell lines were implanted i.b. or s.c. in CD2F, immunocompe-tent mice at a 1.0 x IO6 tumor cell inoculum, no tumors were

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  • INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER

    Fig. 7. Anterioposterior chest radiographiecomparison of a normal and a lung tumor-bearing athymic nude mouse. A normal mouseradiograph is depicted on the left, and a mouse19 days status post-i.b. implantation with 1.0X IO6 NCI-H358 tumor cells is shown on theright. A large tumor involving the right lungand mediastinum is easily identified (arrows)and accounts for the volume loss in the leftlung field. Radiographs were obtained usingthe techniques described in "Materials andMethods."

    Fig. 8. Light micrograph of a large NCI-H460 tumor following i.b. implantation. This represents a paraffin-embedded histology section of a large tumorlocated in the right lung of a nude mouse 19 days following i.b. implantation with1.0 X IO6 NCI-H460 tumor cells. The tumor histology is characteristic of thelarge cell undifferentiated lung carcinoma cell line from which it was derived. H& E, X 500.

    observed at necropsy after 160 days in either the lungs or theskin (n = 10 for the number of animals implanted i.b. or s.c.for each cell line).

    Enzymatically dissociated, fresh human lung cancer specimens, obtained at the time of diagnostic thoracotomy, werealso successfully propagated using the i.b. technique at a 1.0 x10* tumor cell inoculum. Approximately 35% (10 of 29) of the

    fresh primary lung tumors and 66% (2 of 3) of the tumorsmetastatic to the lung were successfully propagated in vivo bythis technique, whereas only 20% (1 of 5) of these same tumorswere successfully established and propagated using the s.c.method at a 1.0 x IO7 tumor cell inoculum (Table 1). Primary

    lung tumors which were successfully propagated i.b. were representative of all 4 major lung tumor histológica! cell types aswell as one relatively rare human pulmonary tumor, a bron-chioloalveolar cell carcinoma. Metastatic lung tumors whichwere successfully propagated i.b. included a transitional cellcarcinoma of the urinary bladder as well as an adenocarcinomaof the prostate (Table 1). Tumors were easily passaged i.b. usingthe enzymatic dissociation technique described in "Materialsand Methods."

    The tumors which were propagated in the mouse i.b. were

    remarkably similar in histológica! appearance to the originalhuman lung tumor specimens from which they were derived.This is exemplified by comparison of the histológica! appearance of a superficially invasive squamous cell carcinoma with aprominent endobronchial exophytic component after its removal from the patient's lung and its appearance after being

    propagated i.b. in the lung of an athymic nude mouse (Fig. 9).Note the squamous cell characteristics in both tumor specimensas well as the striking endobronchial polypoid growth of thetumor propagated i.b. in the right lung of the nude mouse.

    DISCUSSION

    This paper describes a novel intrapulmonary model for theorthotopic implantation and propagation of human lung cancers in athymic nude mice. The procedure is relatively easy toperform, is reproducible, and requires a small number of animals to obtain adequate tumor tissue for experimentation. Themodel uses human lung tumor material rather than less idealcarcinogen-induced animal tumors (5-11) or other non humanlung tumors (30) and potentially affords investigators with animproved opportunity for the study of human lung cancer.

    The present i.b. studies support the concept of the importanceof organ site-specific tumor implantation for optimal tumorgrowth which was originally proposed by Paget (21) and subsequently supported by numerous other studies using metastatictumor models (for review, see Ref. 12) as well as orthotopicmodels for certain human cancers (12, 16, 20, 22-24). Positiveand/or negative selection factors which are present in variousmouse tissues may be major determinants of the success orfailure of human tumor cells to propagate in vivo (for discussion,see Ref. 12). These factors are probably relevant for bothestablished human lung tumor cell lines as well as for freshhuman lung tumor suspensions implanted orthotopically inathymic nude mice. Many tissue characteristics, including theabundant blood supply in the lung, the relatively high compliance of lung tissue, and the availability of numerous endogenousfactors in the pulmonary microenvironment (31-37), conceivably might be responsible for the enhanced survival and growthof tumor cells in the immunodeficient mouse lung. This isfurther supported by the observation that lung tumor cellsretained their original histological characteristics when theywere grown i.b. It is also supported by the reduced lung tumorcell inoculum requirement which was observed for the i.b. modelas compared to the previously described s.c. model which requires a tumor cell inoculum of 1.0 x IO7 cells for optimal

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  • INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER

    tumor development (12-15), and many of these previous s.c.studies have used human tumor fragments which usually contain >1.0 x IO7cells/inoculum. The lower tumor cell inoculum

    requirements for the i.b. model appear consistent with an organsite-specific implantation theory for optimal tumor growth;however, it is important to note that comparably low lungtumor cell inocula have been shown to be effective for propagation of certain small cell lung carcinoma cell lines implantedintracranially in immunodeficient rodents (38, 39). Additionali.b. studies using nonpulmonary human tumors will be necessary to further evaluate this organ-site specificity concept.

    In addition to its utility for propagating continuous long-term human lung tumor cell lines, the intrapulmonary modelalso provides the basis for orthotopic propagation of enzymat-

    ically dissociated fresh human lung tumor tissue in athymicnude mice. This might offer a useful avenue for clinical investigators to expand relatively small surgically excised biopsyspecimens into a tumor volume of sufficient size to providefresh tumor tissue for biochemical, histopathological, and cellbiology studies. This procedure might also allow for propagation and expansion of lung tumor tissue from those patientswho are not candidates for surgical resection, but who undergodiagnostic procedures such as bronchoscopy or needle biopsywhich yield small tumor samples. Since 70 to 80% of all lungcancer patients are inoperable at the time of diagnosis (40,41),this would allow potential access to a much larger lung cancerpatient population for a variety of clinical research and/ordiagnostic studies.

    The intrapulmonary model also offers a potentially attractivesystem for in vivo testing and experimental therapeutics of newpotential antitumor agents. This model might provide phar-macokinetic and pharmacodynamic features more characteristic of human lung cancer than s.c. or intrarenal implantedtumors. In addition, it frequently demonstrates local medias-tinal invasion which is characteristic of certain human lungcancers (41). It also allows for the experimental use of a lethalityend point which might be particularly convenient for large scaledrug-testing studies.

    In addition, noninvasive X-ray procedures can be used toperiodically monitor lung tumor size, and this is especiallyattractive for experimental therapeutics studies. Implantationof tumors in the right lung of nude mice is particularly advantageous, since no other major anatomical structures (e.g., theheart) are located in this area which might interfere with theradiographie evaluation of small human tumor xenografts.Thus, by using both anterioposterior (or posterioanterior) andright lateral radiographie views, a semiquantitative, three-dimensional, radiographie approximation of tumor size can beobtained (Figs. 2 and 7). Such technology could ultimatelyprovide an orthotopic in vivo drug-testing model which closelyresembles the clinical setting, in that it makes possible accurate,periodic, noninvasive monitoring of human lung tumors in ananimal host following treatment with different experimentaltherapeutic modalities.

    In summary, this intrapulmonary model has several advantages over other currently available models and should be ofparticular value for future studies of lung cancer biology andtreatment. Advantages include the following, (a) The procedureprovides the first short-term, reproducible, orthotopic in vivoanimal model for the study of human lung cancer, (b) Lungtumor cells are implanted at the organ site of tumor origin, (c)The i.b. procedure is relatively easy to perform in a modifiedconventional laboratory setting and uses a smaller tumor cellinoculum than that required by other models, (d) It allows for

    propagation of fresh human lung tumor specimens, (e) It potentially provides a suitable in vivo model for the study of humanlung tumor biology, and (/) it offers a potentially attractiveand relevant model for in vivo testing of the efficacy of potentialtherapeutic agents for the treatment of lung cancer.

    ACKNOWLEDGMENTS

    The authors express their appreciation to Dr. J. Minna and Dr. A.Gazdar, NCI Navy Oncology Branch, Bethesda, MD, for providingtumor lines; to Dr. I. J. Fidler, M.D. Anderson Hospital and TumorInstitute, Houston, TX, for his helpful advice regarding this manuscript;and to Kathy Gill for the typing and organization of this text forpublication.

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