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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 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 impeded by the lack of optimal animal models for use in these investigations (4). Animal models which use carcinogens to A major impediment to the study of human lung cancer pathophysiolinduce primary pulmonary tumors have been developed, but ogy, as well as to the discovery and development of new specific antitumor they have not been satisfactory for routine lung cancer studies agents for the treatment of lung cancer, has been the lack of appropriate (5-11). Major disadvantages of the carcinogen-induced animal experimental animal models. This paper describes a new model for the models for study of primary pulmonary tumors include: they propagation of human lung tumor cells in the bronchioloalveolar regions are time consuming (requiring a minimum of 6 mo for comple of the right lungs of athymic NCr-nu/nu mice via an intrabronchial (i.b.) tion); and most importantly, they yield a variety of different implantation procedure. Over 1000 i.b. implantations have been per histological tumor cell types which might not be directly rele formed to date, each requiring 3 to 5 min for completion and having a surgery-related mortality of approximately 5%. The model was used vant to human lung cancer, and thereby limit their usefulness for meaningful biochemical, cell biology, and drug-testing stud successfully for the orthotopic propagation of four established human ies (5-10). lung cancer cell lines including: an adenosquamous cell carcinoma (NCIHI 25); an adenocarcinoma (A549); a large cell undifferentiated carci Athymic nude mouse models have recently been used for the noma (NCI-H460), and a bronchioloalveolar cell carcinoma (NCI-H358). in vivo propagation and study of human pulmonary cancers. When each of the four cell lines was implanted i.b. using a 1.0 x 10*' The predominant prototypes which have been used for these tumor cell inoculum, 100 ±0% (SD) tumor-related mortality was ob studies are the s.c. xenograft (12-16) and the subrenal capsule served within 9 to 61 days. In contrast, when the conventional s.c. method (17-20) models. While these offer rapid and relatively simple for implantation was used at the same tumor cell inoculum, only minimal methods for in vivo propagation of human lung tumors, they (2.5 ±5%) tumor-related mortality was observed within 140 days (P < have limitations, particularly in their use for the study of lung 0.001). Similarly, when a 1.0 x IO5or 1.0 x Ml4cell inoculum was used, cancer biology as well as for the discovery and development of a dose-dependent, tumor-related mortality was observed when cells were effective treatment modalities for human pulmonary malignan implanted i.b. (56 ±24% or 25 ±17%) as compared with the s.c. method cies (12, 15, 16, 20). (5 ±5.7% or 0.0 ±0%) (P < 0.02 and P < 0.05, respectively). Most A potential disadvantage of both the s.c. and subrenal capsule (>90%) of the lung tumors propagated by i.b. implantation were localized models for the propagation and study of human primary pul to the right lung fields as documented by necropsy and/or high-resolution monary malignancies is that tumor cells are not orthotopically chest roentgenography techniques which were developed for these studies. The intrapulmonar}- model was also used for establishment and propa implanted (i.e., tumor cells are not implanted in the lung). Paget originally proposed that human tumor cell populations gation of xenografts derived directly from enzymatically digested, fresh require organ site-specific interaction for optimal maintenance human lung tumor specimens obtained at the time of diagnostic thoraand progression (21). This concept has been widely supported cotomy and representing all four major lung cancer cell types as well as a bronchioloalveolar cell carcinoma. Approximately 35% (10 of 29) of by numerous studies using metastatic tumor models (for review, the fresh primary human lung tumor specimens and 66% (2 of 3) of see Ref. 12) and by recent athymic nude mouse models which tumors metastatic to the lung were successfully propagated i.b. at a 1.0 have been used to study the orthotopic propagation of selected x lit1'tumor cell inoculum, whereas only 20% (1 of 5) of the specimens human solid tumors, such as renal cell carcinoma (12, 16, 20, were successfully grown in vivo via the s.c. route from a 1.0 x 10" tumor 22), pancreatic carcinoma (23), and certain brain tumors (24). cell inoculum. Our experience with this in vivo intrapulmonar} model for A similar approach for the propagation of human lung neo the orthotopic propagation of human lung tumor cells is consistent with plasms in the lungs of athymic nude mice would appear to offer the view that organ-specific in vivo implantation of human tumors facil a potentially very practical and relevant model for the study of itates optimal tumor growth. This new in vivo lung cancer model may this disease. This paper describes the successful development substantially facilitate future studies of the biology and therapeutics of of a novel intrapulmonary model for the orthotopic propagation this catastrophic disease. of human lung cancer cells in athymic nude mice using an i.b.2 implantation technique. INTRODUCTION ABSTRACT 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 nonsurgical 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. 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 homozygotic 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 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research. INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER Table 1 Histopathological characterization of surgically resected human lung tumor specimens propagated by i.b. or s.c. implantation typePrimary*Squamous Cell 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. Cells were maintained at 37°Cin a humidified cabinet in an atmosphere of im im propa propa plantedS.C."21101050005No. planted 5% CÃœ2in air. Cell monolayers approaching gated S.C.*0000101(20)0001(20) i.b."12355132921332No. gated i.b.*33111110 75% confluency were harvested using the DeLarco formulation of trypsin-EDTA and were subcultured for a maximum of 8 to 12 serial passages. For i.b. or s.c. implantation experiments, cells were isolated by centrifugation at 500 x g for 15 min, washed 3 times in 0.9% saline, suspended in a final volume of HBSS, and adjusted to appropriate inoculation densities. All cell lines were documented to be of human origin by isoenzyme and karyotype analyses and were verified to be free of Mycoplasma as well as pathogenic mouse viruses at the time of study. Preparation of Fresh Human Lung Tumors. Fresh human lung tumor specimens obtained at the time of diagnostic thoracotomy were minced into 0.5- to 1.0-mm3 pieces and enzymatically digested as previously cell''AdenocarcinomaSmall cellLarge cellLarge cellBronchioloalveolarSubtotalMetastaticiAdenocarcinoma(prostate)Transitional cell-small (35)'112(66)12 cell (uri bladder)SubtotalTotalNo. nary (38)No. " Fresh human tumor specimens were enzymatically digested and then im planted into athymic nude mice i.b. at a 1.0 x 10' tumor cell inoculum or s.c. at a 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 implan tation 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 the diagram represents the caudal lobe of the right lung, the area where the majority of tumor cells are localized following i.b. implantation. tumor specimens by the NCI-Navy Medical Oncology Branch using standard procedures (25), cultivated, characterized under a variety of growth conditions (26), and kindly provided to the NCI-Developmental Therapeutics Program by Dr. J. Minna and Dr. A. Gazdar. The A549 cell line is a nude mouse ascites-passaged cell line derived at NCIFCRF from the A549 cell line initially described by Giard et al. (27). The cell lines used for this study were propagated in vitro utilizing conventional sterile culture techniques after recovery from cryopreserved seed stock prepared and maintained at the NCI-FCRF tumor repository. Tumor cells were thawed under sterile conditions, washed 3 times with 0.9% saline, and counted with a hemocytometer; cell counts were corrected for viability by the trypan blue dye exclusion method. Cells were cultivated in T75 cm2 flasks in conventional culture medium, consisting of RPMI 1640 (Quality Biologica Is. Gaithersburg, described (28). Cell number, viability, as well as yield were then deter mined, and i.b. or s.c. implantations were performed as described below. All lung tumor diagnoses were histologically confirmed and are sum marized in Table 1. Experimental Animals. Pathogen-free, female NCr-nu/nu mice or BALB/c x DBA/2 F, (hereafter called CD2Fi) immunocompetent mice, approximately 4 to 6 wk of age, were used. All procedures involving the animals were performed in a pathogen-free barrier facility at NCI-FCRF. Implantation i.b. Animals were anesthetized in a Harvard small animal anesthesia chamber (Harvard Biosciences, Boston, MA) at tached to a standard Sonnifer anesthesiology apparatus (Richmond Veterinarian Supply Co., Richmond, VA) using a 7% flow of nebulized Metafane (Pitman Moore, Inc., Washington Crossing, NJ)/100% ox ygen mixture. A 1.0-cm ventral midline incision was made in the neck over the region of the trachea superior to the supraclavicular notch. The surgical procedures were facilitated by the use of a binocular x7 to 10 magnification headset. The glandular tissue surrounding the trachea was separated by blunt dissection, and the trachea was exposed by dissection of the peritracheal muscle sheath. The tissue between the trachea! rings was then incised with the bevel of a 25-gauge needle, and a modified, 27-gauge, blunt-ended 1.2-cm needle was inserted into the trachea and advanced into the right mainstem bronchus (Fig. 1). An inoculum 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 mainstem bronchus. Animals were maintained in a reverse Trendelenberg maneu ver during the inoculation to ensure that the fluid containing the cells remained in the distal airways of the right lung. The skin incision was then closed with surgical skin clips, and the animals were allowed to recover under heat lamps for approximately 10 min, were subsequently returned to their holding cages, and were followed for clinical evidence of pulmonary tumors. Implantation s.c. Tumor cells were implanted at a 1.0 x IO7, 1.0 x IO6, 1.0 x 10s, or 1.0 x 10* inoculum using the previously described s.c. implantation techniques (12-14) in a 0.1-ml final volume of HBSS. Chest Roentgenographic Studies. A Senographe 500T mammo graphie 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., Tren ton, NJ) was used for roentgenographic studies. (The 0.1-mm focal spot is essential for obtaining radiographs with high quality bone and soft tissue detail). Gastrovist radiopaque contrast material (Berlex Industries, Wayne, NJ) was used for contrast dye-enhanced radiographic studies to demonstrate localization of i.b.-implanted material to the various lung fields. The standard i.b. implantation method (described above) was used for these studies with a 0.1-ml volume of the contrast material. Animals were then immediately evaluated by anterioposterior and right lateral chest roentgenography. Lung Tumor Cell Autoradiography Studies. Monolayer cultures of NCI-H460 tumor cells were incubated with medium containing 10 mCi/ml [3H]thymidine (specific activity, 18.2 Ci/mmol; New England Nuclear, Boston, MA) for 48 h prior to implantation. An inoculum of 1.0 x IO6tumor cells was suspended in a 0.1-ml final volume of HBSS and then administered i.b. to NCr-nu/nu mice as described above. Control, radiolabeled NCI-H460 cells were incubated on glass cover- 5133 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research. INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER Fig. 2. Demonstration of localization of i.b.-implanted material to the right lung by contrast dye-enhanced radiography. The chest radiograph on the left represents an anterioposterior view, and the one on the right, a right lateral view, of a mouse immediately following i.b. implantation with radiopaque dye. Note the localization of the contrast material to the caudal (lower) lobe of the right lung (arrows). This corresponds to the highlighted area in Fig. 1 representing the right lower lobe. Radio graphs were performed as described in "Ma terials and Methods." -*- l'ß V Fig. 3. Localization of tumor cells to the bronchioloalveolar regions of the caudal lobe of the right lung following i.b. implantation. This light micrograph illustrates a par affin-embedded, hematoxylin- and eosin-stained histology section of the caudal lobe of the right lung of an athymic nude mouse l h post-i.b. implantation with 1.0 x 10" NCIH460 tumor cells. A representative alveolus (a) and airway (o) are labeled for comparison. Note the clustering of tumor cells in the bronchioloalveolar regions with filling of the alveolar spaces by the tumor cells. Representative tumor cells are demonstrated by arrows. Normal alveoli are prominent in the lower portion of the micrograph. X300. v • * fi* Fig. 4. Autoradiographic demonstration of radiolabeled tumor cell distribution in the cau dal lobe of the right lung. This light micro graph illustrates a periodic acid-Schiff-stained, hematoxylin-counterstained serial section of the caudal lobe of the right lung l h following i.b. implantation with a 1.0 x IO6 NCI-H460 tumor cell inoculum which was labeled for 48 h prior to implantation with [3H]thymidine as described in "Materials and Methods." Heav ily radiolabeled tumor cells are easily identified in the bronchioloalveolar regions (representa tive cells are demonstrated by arrows). Inset, control radiolabeled cells, placed on a glass coverslip for I h. shown for comparison. A representative alveolus (a) and airway (b) are also labeled in the figure. Normal alveoli are present in the upper left and lower right por tions of the micrograph. slips at 37°Cin the presence of 5% COj for l h and then processed for perfusion with 10% low-molecular-weight dextran (Abbott Lab., Chi autoradiographic evaluation. Mice that received i.b. implants with cago, IL) (w/v) in 0.9% saline followed by 2% glutaraldehyde in 0.1 M radiolabeled cells were anesthetized with 0.1 ml of sodium pentobarbital cacodylate buffer (pH 7.4) as previously described (11). Individual lung (Richmond Veterinary Supply Company, Richmond, VA) l h after the lobes were collected and immersed in the above glutaraldehyde solution i.b. implantation, and lung tissues were then fixed in situ by vascular for 2 h, followed by further fixation in 10% buffered formalin for 2 5134 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research. 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 micrographs demonstrate 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 of the right lower lobe. A, x 500; fi, x 200. days. Glass slides (Fisherbrand; Fisher Scientific, Fairlawn, NJ) were precleaned for 5 min with 70% ethyl alcohol containing 1% hydrochlo ric acid, rinsed well with running distilled water, placed in a 95% ethyl alcohol rinse for S min, and subsequently air dried. Serial 5 ^ in sections of each lung lobe were placed onto precleaned glass slides at three levels approximately 100 urn apart. Duplicate sections were taken at each level. The fixed lung tissues were subsequently embedded in paraffin, and autoradiography and staining procedures were performed as pre viously described (29). Lung tissue sections were then microscopically evaluated for radiolabeled tumor cell localization. Lung Tumor Cell Localization Studies. Athymic nude mice were implanted i.b. with 1.0 x IO6 NCI-H460 cells, and animals were then Monitoring of Animals for Tumor Development. Animals that received either i.b. or s.c. implants were carefully followed daily for signs of tumor development. Throughout the observation period, animals de veloping respiratory distress were killed by cervical dislocation. All those animals remaining after 140 days were also killed, and the major organs (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 then evaluated for the presence of tumor or other histopathology. Mortality data were based on only those animals that died from direct effects of tumor growth and/or metastatic disease before 140 days. fixed in situ by vascular perfusion as described above at 1 h, 7 and 10 days following implantation. Individual lung lobes were removed and separated, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and microscopically evaluated for tumor cell localization. RESULTS The i.b. implantation method (Fig. 1) provides for the inoc ulation of human lung tumor cell suspensions into the right 5135 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research. INTRAPULMONARY MODEL sc 80 60 40 20 100 140 u..„' 0 20 60 100 * A 0sc. eI£ 20 60 100 140 20 60 100 140 MinnlUU80604020B|..IB"H^^5-+ SC IB 100 80 60 40 20 20 60 100 140 20 60 IB 100 140 SC 100 80 60 40 20 100 140 20 LUNG CANCER sections of individual mouse lung lobes following i.b. implan tation of 1.0 x IO6 NCI-H460 cells demonstrated localization 100 20 FOR HUMAN 60 Days Post Implant Fig. 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 mortality for i.b. and s.c. implantations are summarized in Fig. 6, where A to D represent mortality from NCI-HI25, A549, NCI-H460, and NC1-H358 cell lines, respec tively. »,1.0 X IO6;A, 1.0 x 10s; andO, 1.0 x IO4tumor cell inocula. Each point represents 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 lines at 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 x IO4 tumor cell i.b. inocula, respectively. C and D, n = 20 for all three i.b. tumor cell inocula; n = 10 for the 3 different s.c. tumor cell inocula for the 4 different cell lines tested. The low mortality for s.c. implantation was related to the low tumor propagation efficiencies. of the majority of the tumor cells to the bronchioloalveolar regions of the caudal lobe of the right lung (Fig. 3). Localization of tumor cells to this region of the right lung l h after i.b. implantation was more clearly demonstrated when 1.0 x IO6 radiolabeled NCI-H460 cells were used (Fig. 4). Again, only the caudal lobe of the right lung consistently contained radiolabeled tumor cells when individual lung lobes were separately evaluated. In addition, microscopic tumor nodules were ob served in predominantly the right lower lobe 7 and 10 days following i.b. implantation when individual lung lobes were again evaluated at these longer time intervals (Fig. 5). These different studies all clearly demonstrated that the majority of tumor cells initially resided in the bronchioloalveolar regions of the caudal lobe of the right lung following i.b. implantation. Athymic nude mice implanted i.b. with human lung tumor cells demonstrated clinical symptoms of cachexia and dyspnea as the pulmonary xenografts became progressively larger. These were similar to the symptoms frequently observed in human lung cancer patients. The tumor cells grew most frequently in the peripheral regions of the right lung following i.b. implan tation (Fig. 5); however, certain tumors did grow predominately in the airways (Fig. 9). Implantation i.b. produced tumors that ultimately led to the death of the animals (Fig. 6). Lung tumor progression could also be readily followed roentgenographically (Fig. 7), thus allowing tumor-bearing animals to be monitored noninvasively. When the 251 tumor-bearing animals repre sented in Fig. 6 were studied at necropsy following i.b. implan tation, 91% of the tumors implanted by this method were localized to the right lung. Other tumor locations included the left lung (1%), trachea (2%), or peritracheal (6%) area. Local mediastinal invasion was observed at necropsy in approximately 90% of the tumor-bearing animals following i.b. inoculation. In 3% of the i.b.-implanted, tumor-bearing animals, distant métastaseswere also observed at necropsy in the left lung, lymph nodes, liver, or spleen. The histologies of tumors obtained from the i.b. implantation model were consistently similar to the parent human lung tumor cell lines from the which they were derived (n = 251). As an example, a tumor obtained from the right lung of a nude mouse 19 days following i.b. implantation with the NCI-H460 cell line demonstrated characteristic histopathological features of a hu man large cell undifferentiated carcinoma of the lung (Fig. 8). The reproducibility of this model among different cell lines was demonstrated in that it produced similar pulmonary tumors and death patterns for the 4 lung tumor cell lines tested (Fig. 6). Reproducibility within cell lines was also demonstrated for the NCI-H460 cell line when it was implanted i.b. multiple times over a 6-mo interval (coefficient of variation for mean survival was 24% for this cell line implanted on 4 separate occasions). The intrapulmonar) model was also compared to the conven tional s.c. model for effectiveness in propagation of human lung tumor cells. When the 4 different human lung tumor cell lines were implanted i.b. at 3 different tumor cell inocula, 100% tumor-related mortality was observed at the 1.0 x IO6 tumor cell inoculum, and a dose-dependent response was noted with lower (1.0 x 10s or 1.0 x 10") tumor cell suspensions (Fig. 6). In contrast, minimal (5%) tumor-related mortality was observed within 140 days for the s.c. model for the 4 cell lines at all tumor cell inocula tested (Fig. 6). When tumor cells from the 4 cell lines were implanted i.b. or s.c. in CD2F, immunocompetent mice at a 1.0 x IO6 tumor cell inoculum, no tumors were mainstem bronchus of athymic nude mice. Over 1000 i.b. implants have been performed to date, each requiring an average of 3 to 5 min for completion and having a surgery-related mortality of approximately 5%. This model provides an orthotopic site for tumor implantation in that the majority of tumor cells are deposited in the bronchioloalveolar regions of the right lung, as verified by radiopaque dye-enhanced radiographie stud ies, routine histology, and autoradiographic evaluation (Figs. 2 to 4). Radiopaque dye-enhanced radiographie studies were initially performed, whereby radiopaque contrast material was im planted i.b., and low-dose, high-resolution chest radiographs were then immediately performed. These studies clearly dem onstrated radiographie localization of the radiopaque contrast material predominantly to the caudal (lower) lobe of the right lung (Fig. 2). Similarly, microscopic evaluation of histological 5136 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research. INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER Fig. 7. Anterioposterior chest radiographie comparison of a normal and a lung tumorbearing athymic nude mouse. A normal mouse radiograph is depicted on the left, and a mouse 19 days status post-i.b. implantation with 1.0 X IO6 NCI-H358 tumor cells is shown on the right. A large tumor involving the right lung and mediastinum is easily identified (arrows) and accounts for the volume loss in the left lung field. Radiographs were obtained using the techniques described in "Materials and Methods." remarkably similar in histológica! appearance to the original human lung tumor specimens from which they were derived. This is exemplified by comparison of the histológica! appear ance of a superficially invasive squamous cell carcinoma with a prominent endobronchial exophytic component after its re moval 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 specimens as well as the striking endobronchial polypoid growth of the tumor propagated i.b. in the right lung of the nude mouse. DISCUSSION Fig. 8. Light micrograph of a large NCI-H460 tumor following i.b. implan tation. This represents a paraffin-embedded histology section of a large tumor located in the right lung of a nude mouse 19 days following i.b. implantation with 1.0 X IO6 NCI-H460 tumor cells. The tumor histology is characteristic of the large 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 the skin (n = 10 for the number of animals implanted i.b. or s.c. for each cell line). Enzymatically dissociated, fresh human lung cancer speci mens, obtained at the time of diagnostic thoracotomy, were also successfully propagated using the i.b. technique at a 1.0 x 10* tumor cell inoculum. Approximately 35% (10 of 29) of the fresh primary lung tumors and 66% (2 of 3) of the tumors metastatic to the lung were successfully propagated in vivo by this technique, whereas only 20% (1 of 5) of these same tumors were 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 rep resentative of all 4 major lung tumor histológica! cell types as well as one relatively rare human pulmonary tumor, a bronchioloalveolar cell carcinoma. Metastatic lung tumors which were successfully propagated i.b. included a transitional cell carcinoma of the urinary bladder as well as an adenocarcinoma of the prostate (Table 1). Tumors were easily passaged i.b. using the enzymatic dissociation technique described in "Materials and Methods." The tumors which were propagated in the mouse i.b. were This paper describes a novel intrapulmonary model for the orthotopic implantation and propagation of human lung can cers in athymic nude mice. The procedure is relatively easy to perform, is reproducible, and requires a small number of ani mals to obtain adequate tumor tissue for experimentation. The model uses human lung tumor material rather than less ideal carcinogen-induced animal tumors (5-11) or other non human lung tumors (30) and potentially affords investigators with an improved opportunity for the study of human lung cancer. The present i.b. studies support the concept of the importance of organ site-specific tumor implantation for optimal tumor growth which was originally proposed by Paget (21) and sub sequently supported by numerous other studies using metastatic tumor models (for review, see Ref. 12) as well as orthotopic models for certain human cancers (12, 16, 20, 22-24). Positive and/or negative selection factors which are present in various mouse tissues may be major determinants of the success or failure of human tumor cells to propagate in vivo (for discussion, see Ref. 12). These factors are probably relevant for both established human lung tumor cell lines as well as for fresh human lung tumor suspensions implanted orthotopically in athymic nude mice. Many tissue characteristics, including the abundant blood supply in the lung, the relatively high compli ance of lung tissue, and the availability of numerous endogenous factors in the pulmonary microenvironment (31-37), conceiv ably might be responsible for the enhanced survival and growth of tumor cells in the immunodeficient mouse lung. This is further supported by the observation that lung tumor cells retained their original histological characteristics when they were grown i.b. It is also supported by the reduced lung tumor cell inoculum requirement which was observed for the i.b. model as compared to the previously described s.c. model which re quires a tumor cell inoculum of 1.0 x IO7 cells for optimal 5137 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research. INTRAPULMONARY MODEL FOR HUMAN LUNG CANCER f Hi g. g £• 8~.g eS. t ë'f • o — a g 2 =* r = |t•M I fis çao .i«ü_ * ' S. 5Õ l SI i-^MI *3lf|s •¿à g =1« ii-JH ihm £§i-H-M-S 's l3 «"s gif<" if'Zall-n S E Jlfllj S|s«-il "¡13 lliJil II' i *•-ri >> tu E o n •S^s| E «>-^>2 g ¿•a3=l^ ¿Hilf ¡Itili c M,¿« «5 ob ^ ^ 'C ^ * o «•£ «Z a-o o « Si OD u T3 focII1 Õ M« £Sg 2 £00»1 OO.'- — .a 5 -C J= * &.i su ¡li; &•? § ° si •S1 E & 5c k 2 •-* ' : " •$ "2 • ¡È«!« »'S o-T M > 2P -2 5138 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research. \o X 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 con tain >1.0 x IO7cells/inoculum. The lower tumor cell inoculum requirements for the i.b. model appear consistent with an organ site-specific implantation theory for optimal tumor growth; however, it is important to note that comparably low lung tumor cell inocula have been shown to be effective for propa gation of certain small cell lung carcinoma cell lines implanted intracranially in immunodeficient rodents (38, 39). Additional i.b. studies using nonpulmonary human tumors will be neces sary to further evaluate this organ-site specificity concept. In addition to its utility for propagating continuous longterm human lung tumor cell lines, the intrapulmonary model also provides the basis for orthotopic propagation of enzymatically dissociated fresh human lung tumor tissue in athymic nude mice. This might offer a useful avenue for clinical inves tigators to expand relatively small surgically excised biopsy specimens into a tumor volume of sufficient size to provide fresh tumor tissue for biochemical, histopathological, and cell biology studies. This procedure might also allow for propaga tion and expansion of lung tumor tissue from those patients who are not candidates for surgical resection, but who undergo diagnostic procedures such as bronchoscopy or needle biopsy which yield small tumor samples. Since 70 to 80% of all lung cancer patients are inoperable at the time of diagnosis (40,41), this would allow potential access to a much larger lung cancer patient population for a variety of clinical research and/or diagnostic studies. The intrapulmonary model also offers a potentially attractive system for in vivo testing and experimental therapeutics of new potential antitumor agents. This model might provide pharmacokinetic and pharmacodynamic features more characteris tic of human lung cancer than s.c. or intrarenal implanted tumors. In addition, it frequently demonstrates local mediastinal invasion which is characteristic of certain human lung cancers (41). It also allows for the experimental use of a lethality end point which might be particularly convenient for large scale drug-testing studies. In addition, noninvasive X-ray procedures can be used to periodically monitor lung tumor size, and this is especially attractive for experimental therapeutics studies. Implantation of tumors in the right lung of nude mice is particularly advan tageous, since no other major anatomical structures (e.g., the heart) are located in this area which might interfere with the radiographie evaluation of small human tumor xenografts. Thus, by using both anterioposterior (or posterioanterior) and right lateral radiographie views, a semiquantitative, three-di mensional, radiographie approximation of tumor size can be obtained (Figs. 2 and 7). Such technology could ultimately provide an orthotopic in vivo drug-testing model which closely resembles the clinical setting, in that it makes possible accurate, periodic, noninvasive monitoring of human lung tumors in an animal host following treatment with different experimental therapeutic modalities. In summary, this intrapulmonary model has several advan tages over other currently available models and should be of particular value for future studies of lung cancer biology and treatment. Advantages include the following, (a) The procedure provides the first short-term, reproducible, orthotopic in vivo animal model for the study of human lung cancer, (b) Lung tumor cells are implanted at the organ site of tumor origin, (c) The i.b. procedure is relatively easy to perform in a modified conventional laboratory setting and uses a smaller tumor cell inoculum than that required by other models, (d) It allows for propagation of fresh human lung tumor specimens, (e) It poten tially provides a suitable in vivo model for the study of human lung tumor biology, and (/) it offers a potentially attractive and relevant model for in vivo testing of the efficacy of potential therapeutic 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 providing tumor lines; to Dr. I. J. 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Martin, and J. D. Wilson (eds.), Harrison's Principals of Internal Medicine, pp. 1572-1580. New York: McGraw-Hill Book Co., 1983. 5140 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research. Novel Intrapulmonary Model for Orthotopic Propagation of Human Lung Cancers in Athymic Nude Mice Theodore L. McLemore, Mark C. Liu, Penny C. Blacker, et al. Cancer Res 1987;47:5132-5140. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/47/19/5132 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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