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Ann Thorac Surg 2001;71:1120-1125
© 2001 The Society of Thoracic Surgeons

Original article: general thoracic

Validation of an orthotopic model of human lung cancer with regional and systemic metastases

Michael R. Johnston, MDa, John B.M. Mullen, MDb, Marco E. Pagura, BSca, Randy B. Howard, PhDa

a Division of Thoracic Surgery and the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada
b Department of Pathology, Mount Sinai Hospital, University of Toronto, Toronto, Canada

Accepted for publication November 19, 2000.

Address reprint requests to Dr Johnston, Division of Surgical Oncology, Princess Margaret Hospital, 610 University Ave, Suite 3-130, Toronto, Ontario, Canada M5G 2M9
e-mail: michael.johnston{at}uhn.on.ca


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. We developed an orthotopic model of human lung cancer that exhibits highly predictable regional and systemic metastases. This study examines the response of the model when treated with conventional and experimental chemotherapy.

Methods. NCI-H460 tumor fragments were implanted into the right caudal lung lobe of a nude rat. Treatment commenced 2 weeks later. We assessed response by comparing primary tumor and mediastinal lymph node weights, total body weight, and length of survival with untreated, tumor-bearing control animals. We also calculated the incidence of metastasis to kidney, bone, brain, and contralateral lung in treated versus untreated animals.

Results. Mitomycin and cisplatin showed broad activity against primary and metastatic disease. The matrix metalloproteinase inhibitor batimastat, low-dose cisplatin, and mitomycin significantly prolonged survival. High-dose cisplatin caused renal toxicity that shortened survival. Brain metastases did not respond to mitomycin, consistent with its poor blood-brain barrier penetration.

Conclusions. Responses were similar to NCI-H460 in vitro data and consistent with clinical experience for these drugs. Drug-related toxicities similar to those seen in clinical practice were detected.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Lung cancer is the leading cause of cancer deaths in North America. Most patients die of progressive metastatic disease despite aggressive local and systemic therapies. Development of new or improved therapeutic agents is a high priority. To screen these agents, the National Cancer Institute (NCI) has set up a drug discovery program that entails initial in vitro assessment of activity against a panel of human cancer cell lines [1]. Active drugs are then passed to in vivo systems modeled after particular diseases.

The subcutaneous nude mouse xenograft model is widely used for in vivo screening. It provides information on primary tumor response, but because these subcutaneously implanted tumors rarely express invasive or metastatic phenotypes, invasion and metastasis cannot usually be evaluated. In recent years, orthotopic models have been devised in which human tumors are implanted directly into the appropriate organ or site of origin in the laboratory animal. Thus, breast cancer grows in the breast and colon cancer in the colon instead of in the subcutaneous tissue of the mouse or rat. The ensuing tumors often exhibit both invasive and metastatic behavior [2–8].

We have developed and characterized an orthotopic lung cancer model in nude rats using an endobronchial implantation technique that produces a growth and metastatic pattern highly consistent with human lung cancer. A primary lung tumor grows in the right caudal lobe bronchus, invades into lung parenchyma, and then spreads to mediastinal lymph nodes. Distant metastatic disease subsequently develops at multiple sites, including bone, brain, kidney, and opposite lung [9]. The untreated animal dies of progressive cachexia within 5 weeks of implantation.

The purpose of this study was to characterize the response of the model when tumor-bearing animals are treated with one of four chemotherapy agents: doxorubicin, mitomycin, cisplatin, and the novel matrix metalloproteinase inhibitor, batimastat. Both metastatic spread and physical end points, such as length of survival, body weight, and tumor weight, were used to evaluate drug performance. The responses observed were consistent with clinical experience when these agents are used in patients with non–small cell lung cancer, providing a first step in the validation of this preclinical model.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Animals
Male nude rats (CR:NIH-RNU; Charles River Inc, Frederick Cancer Research Facility, Frederick, MD) were received at 4 weeks of age and acclimated for 1 to 2 weeks before entering study protocols. Rats were kept in sterilized cages and fed autoclaved food and water ad libitum. Manipulations were done under sterile conditions in a laminar flow hood. All studies were approved by the Samuel Lunenfeld Research Institute Animal Care Committee. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health.

Cell implantation for donor tumors
NCI-H460 human lung large cell carcinoma cells were grown in T80 flasks in RPMI 1640 containing 5% fetal calf serum. The H460 cells were harvested by trypsinization and adjusted to a final concentration of 106 cells/80 µL for endobronchial implantation. On the morning of implantation, 5-week-old males were irradiated with 500 rad of whole-body gamma irradiation from a 127Cs source at 120 rad/min. Rats were anesthetized with ketamine/xylazine (110 and 12 mg/kg, C.D.M.V. Inc, Guelph, ON) given by intramuscular injection, then implanted with 106 tumor cells using a 20-gauge, 2-inch-long Teflon catheter passed into the right caudal lobe via a small tracheotomy incision as previously described [10]. The tracheotomy was repaired with a 6-0 Proline suture (Ethicon Inc, Sommerville, NJ) and the incision closed with sterile wound clips. The rats were placed back into their cages on sterile rolled drapes to maintain them in a semi-upright position.

Implantation of tumor fragments
Tumor-bearing animals were sacrificed by CO2 asphyxiation at approximately 5 weeks after implantation and the tumors harvested in cold RPMI 1640. A 0.5- to 1.0-g portion of viable tumor was removed and mechanically cut into 1- to 2-mm-diameter pieces by "crossed scalpels" technique in a sterile Petri dish. The pieces were then mixed thoroughly and divided into 50-mg portions. Each 50-mg portion was loaded into a 16-gauge, 2-inch-long Teflon catheter [9]. Six-week-old male nude rats were then implanted with tumor fragments. Irradiation, anesthesia, and implantation procedures were similar to those used for cultured tumor cell implantation. Animals were treated postimplantation with Augmentin (Beecham Labs, Bristol, TN) at 0.35 mg/mL in the drinking water for 2 weeks.

Preparation of agents
Three conventional drugs and one novel drug were selected for this investigation. Doxorubicin, cisplatin, and mitomycin were obtained from the hospital pharmacy (Bristol Myers Canada, Adria Laboratories Canada). British Biotechnology Ltd, (Oxford, UK) kindly provided batimastat. All agents were prepared according to manufacturers’ instructions. Doxorubicin was dissolved in sterile isotonic saline (0.9%) and cisplatin and mitomycin were dissolved in sterile water. Batimastat was dissolved in isotonic saline with Tween 80 (1% v/v), vortexed, placed in an ultrasonic waterbath for 20 minutes, then treated with an ultrasonic probe for 5 minutes until a uniform suspension was achieved. Batimastat, doxorubicin, and mitomycin were stored at 4°C and cisplatin was stored at room temperature. Doxorubicin, cisplatin, and mitomycin were wrapped with aluminum foil to protect the contents from light. Batimastat was resonicated for 10 minutes each day to ensure homogeneity of the suspension before administration. All agents were administered via intraperitoneal (IP) injection in a volume of 5 mL/kg of the animal’s body weight. The animals in each experiment were weighed before implantation and divided into treatment and control groups to insure no more than 10% mean weight difference between groups.

Four drugs were tested: doxorubicin (5 mg/kg/week), batimastat (75 mg/kg/day), cisplatin at two dosages (5 and 6 mg/kg/week), and mitomycin (2 mg/kg/week). Drug therapy commenced 14 days after implantation. The endobronchial tumor at this time is approximately 200 to 300 mg in size and there is no demonstrable evidence of metastasis [9]. Animals were euthanized when they showed signs of significant morbidity or impending death. At necropsy, the primary tumor in the right lung was easily recognized, as was tumor in the anterior mediastinal nodal group and various other metastatic sites (Fig 1). The intact animal, the primary tumor in the right lung, and the mass of anterior mediastinal lymph nodes were weighed fresh immediately after death. To characterize the metastatic pattern, the heart-lung blocks, kidney, brain, and chest wall were fixed in 10% buffered formalin and embedded in paraffin. The primary tumor and mediastinal lymph nodes were cross-sectioned, and the left lung lobe, kidney and brain were serially sectioned. All tissue sections were stained with hemotoxylin and eosin, and examined in a blinded fashion by a pathologist (J.B.M.M.).



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  		Fig 1. Chest cavity of a nude rat containing: (1) right caudal lobe tumor arising from NCI-H460 tumor fragments implanted endobronchially, with (2) regional metastasis to anterior mediastinal lymph nodes and (3) systemic metastasis to rib.

 
Nineteen percent of the animals entered on study died before the initiation of drug dosing at 14 days. Most of these deaths were attributed to anesthetic complications after implantation, but some also died of infection. Six animals (5%) died between 14 and 25 days after implantation and were excluded from the final analysis because the metastatic profile seen in our previous studies with this model is not fully established before day 26. These deaths were equally divided between control and treatment groups.

Control groups
Concurrent control animals were included with each drug group studied. These tumor-bearing animals were injected with the appropriate drug vehicle, using the same schedule as the corresponding drug. Statistical analysis performed on the control animals from the five treatment groups, using an unpaired t test for the primary tumor weight, length of survival, body weight, mediastinal lymph node weight, and a contingency table with Fisher’s exact test for the metastatic pattern, showed no significant difference between control groups. For purposes of reporting, the control animals were therefore grouped together.

Statistical analysis
Statistical analysis of survival, body weight, primary tumor weight, mediastinal lymph node weight, and incidence of metastasis was performed on a Macintosh Performa 5200 computer using Statview (Version 4.1; Abacus Concepts, Berkeley, CA) statistical software. These end points were evaluated using either a contingency table with a Fisher’s exact test or a one-way analysis of variance with a Fisher’s post hoc test. In all cases, p less than or equal to 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
In Figure 1, the gross anatomic appearance of the primary and metastatic NCI-H460 tumor in the nude rat is shown at necropsy. The weights of the primary lung tumor and the anterior mediastinal lymph node group (almost completely replaced by tumor) are depicted graphically in Figure 2 for the various treatment arms. Figure 3 shows the effects of drug treatment on length of survival and body weight at death.



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  		Fig 2. Primary tumor and mediastinal lymph node weight at death. *p < 0.05 compared with controls; **p < 0.01 compared with controls; +p < 0.01 compared with CDDP 5 mg. (BAT = batimastat; DOX = doxorubicin; CDDP = cisplatin; MMC = mitomycin C.)

 


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  		Fig 3. Length of survival and final body weight after drug treatment. *p < 0.05 compared with controls; **p < 0.01 compared with controls; +p < 0.01 compared with CDDP 5 mg. (BAT = batimastat; DOX = doxorubicin; CDDP = cisplatin; MMC = mitomycin C.)

 
Treatment with doxorubicin, mitomycin, and both dosages of cisplatin resulted in a decrease in tumor weight at the primary site and in the mediastinal lymph nodes (Figure 2). All three groups lost significantly more body weight than control animals (Fig 3). Batimastat therapy resulted in a borderline significant decrease (p = 0.06) in lymph node weight, but not primary tumor weight.

Histological examination of the kidneys in the higher dose cisplatin group showed evidence of renal injury consistent with platinum toxicity. This included ductal tubular dilatation, rare epithelial cell necrosis, and cellular atypia with giant hyperchromatic nuclei and prominent nucleoli (Fig 4).



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  		Fig 4. Light micrograph of kidney from a nude rat treated with cisplatin (6 mg/kg/week). Evidence of nephrotoxicity includes: (1) distal tubular dilation, (2) epithelial cell necrosis, (3) cellular atypia with giant nuclei, and (4) prominent nucleoli (H & E stain, 300x).

 
Table 1 shows the percent incidence of metastases at regional and systemic sites after drug treatment compared with tumor-bearing control animals. Doxorubicin and batimastat therapy showed no reduction in incidence at any site and perhaps an increased incidence in the brain with batimastat. In distinction, both dosages of cisplatin and mitomycin showed marked reduction in incidence at most regional and systemic sites. A notable exception was the lack of response with mitomycin in the brain.


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  		Table 1. Percent Incidence of Metastases at Death in Treated and Control Animals

 
The net outcome of drug therapy resulted in a significant survival advantage only in the batimastat, the lower dose cisplatin, and the mitomycin groups (Fig 3).


   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Recently, we described an orthotopic model system of human lung cancer in the nude rat consisting of a primary lung tumor, regional metastasis to mediastinal lymph nodes, and systemic metastases in kidney, brain, bone, and lung [9]. The present study characterized the clinical response of this model system when these tumor-bearing animals are treated with one of four chemotherapeutic agents. The model was found to accurately reflect drug sensitivity patterns consistent with non–small cell lung cancer and the particular NCI-H460 cell line used in the model. It was also capable of detecting drug toxicity and the cumulative effects of tumor/drug interactions within the intact organism. These characteristics make the model particularly attractive for screening new anticancer therapies for lung cancer.

The purpose of this study was to validate a clinically relevant model of lung cancer utilizing an extremely aggressive orthotopic tumor from which all animals die of progressive local and widespread metastatic disease within 5 weeks of implantation [9]. In this setting, it is unreasonable to expect complete clearing of tumor from either primary or metastatic sites when treating with a single agent. Thus, we report the incidence of metastatic involvement in drug-treated animals compared with concurrently evaluated, untreated, tumor-bearing animals. Metastatic involvement of individual organs was graded as either present or absent, a far more stringent method than counting metastases or calculating tumor volumes.

We evaluated four drugs, each given systemically as a single agent over a period of at least 3 weeks. The drugs included the standard cytotoxic agents, doxorubicin, cisplatin, and mitomycin, and the experimental matrix metalloproteinase inhibitor, batimastat. All three standard agents have a long history of use in lung cancer therapy and all three have been shown to exhibit activity against the NCI-H460 large cell carcinoma cell line either in vitro or in vivo [11–13]. Although in the analogous clinical setting none of these drugs would normally be given as a single agent, we chose to administer the drugs individually to more easily assess responsiveness of the model system in terms of both tumoricidal activity and systemic toxicity.

This study was not intended to assess drug efficacy, per se, but rather to gain useful information about the utility of the model system. Nonetheless, each drug was given at, or as close to, the maximum tolerated dose as possible. Philips and associates described the dose-related toxic effects of mitomycin in rats [14] and found the intraperitoneal LD50 to be 2.5 mg/kg given as a single intraperitoneal dose. Because our animals were treated weekly and received between two and four doses, we chose to lower the dose to 2 mg/kg. These animals showed a highly significant decrease in the weights of the primary tumor and mediastinal lymph node metastasis. Mitomycin also profoundly lowered the incidence of regional and systemic metastases, the only notable exception being its failure to lower the incidence of brain metastasis. However, this is in keeping with the known inability of mitomycin to cross the blood-brain barrier [15, 16].

Our laboratory has had extensive experience with the use of doxorubicin in isolated lung preparations [13, 17], in whole-animal models [18, 19], and in human lung perfusions [20]. Before this study, a dose toxicity experiment was performed with weekly administration of intraperitoneal doxorubicin in the Rowett nude rat. Animals tolerated up to 4 mg/kg without weight loss or distress. At 6 mg/kg, 3 of 4 animals died after 3 weeks, and at 8 mg/kg, 3 of 4 animals died after 2 weeks. For the present study, we chose a doxorubicin dosage of 5 mg/kg, which decreased the weight of the primary tumor but had no demonstrable effect on the incidence of metastasis.

The LD50 for cisplatin in rats is variously reported to be between 4 and 8 mg/kg [21, 22]. Even doses as low as 3 to 4 mg/kg cause significant blood urea nitrogen elevations. We used two dosages of cisplatin in this study, 5 and 6 mg/kg/week, in order to ensure that the maximum tolerated dose was given to these tumor-bearing animals. Although both dosages showed highly significant tumoricidal effects, with significant decreases in both tumor and lymph node weights and in the incidence of metastasis, the higher dosage was superior to the lower dosage at all sites. Nevertheless, the lower dosage animals weighed more and lived longer then the higher dosage group. These differences are most likely related to histologically confirmed cisplatin renal toxicity in the higher dosage group (Fig 4). No serum creatinine or urea nitrogen levels were obtained in this study.

Because most patients with lung cancer die of systemic metastasis, an antimetastatic agent is especially appealing in this disease. Batimastat has been shown to decrease the incidence of metastases in human colon carcinoma, rat mammary carcinoma, and orthotopic human liver cancer model systems [23–26]. It is presently being evaluated in clinical trials. In both animal and clinical studies, the drug is well tolerated without major toxicity. In a pilot study using batimastat at 50 mg/kg/day and in the present study with 75 mg/kg/day, we found the drug to be well tolerated with no evidence of systemic toxicity.

The capability of assessing multiple end points appears to improve the sensitivity of the model system to demonstrate a treatment effect. By describing patterns of response in a model system, the results may also suggest mechanisms of action or biological properties of a particular agent. For instance, when survival was measured, batimastat was found to significantly increase length of survival despite the fact it showed no consistent effect on tumor size or on the incidence of metastases. Thus, the drug may be slowing, but not eradicating, the metastatic process. A study by Sledge and associates supports this notion. They showed that batimastat was more effective in inhibiting local-regional tumor recurrence and formation of metastases if given as adjuvant therapy after primary tumor resection [27]. The activity of doxorubicin in the model is another example. This drug significantly decreased primary lung tumor weight both in this study and in a pilot study in which a lower dose (4 mg/kg) was given (2.72 ± 0.30 vs 4.79 ± 0.41 g in control animals). It even decreased the weight of tumor in mediastinal nodes, but failed to impact on the incidence of regional or systemic metastases. These results imply that at this dosage of doxorubicin there may be selective resistance to clonal elements within the tumor that express the metastatic phenotype.

Our model was also found to reflect drug toxicity, which in other animal models has been shown to predict human drug toxicity [28]. Both cisplatin- and mitomycin-treated animals showed marked primary and metastatic tumor response, but this resulted in an increase in length of survival only in the lower-dose cisplatin and mitomycin groups. With higher-dose cisplatin, animals lived no longer and weighed significantly less at death than untreated, tumor-bearing rats. At necropsy, their kidneys showed histological evidence of renal tubular injury (Fig 4). Although no biochemical parameters of renal function were measured, thorough necropsies of the animals failed to uncover any other cause for their decreased survival. This type of complex drug/tumor/organism interaction, which is so common in clinical medicine, can rarely be modeled experimentally.

Creation of this model system entails implantation of tumor fragments by endobronchial implantation through a small tracheotomy. Multiple studies have now confirmed a near 100% tumor take rate in the right caudal lobe with the NCI-H460 cell line [9, 11, 13, 17]. In this study, 82% of control animals also developed distinct tumor masses in the left lung. We do not know whether these left lung tumors signify systemic metastases, as occurs in brain, bone, and kidney, or whether they represent endobronchial spread from spillage of tumor cells at the time of implantation [9]. The present study does not clarify this issue, because the active drugs, mitomycin and cisplatin, showed good response against primary tumors, metastatic tumors, and the enigmatic left lung tumors. Future studies with more active antimetastatic agents or molecular analysis of these lesions may elucidate the origin of these tumors.

This model system has both strengths and limitations. Because the primary orthotopic tumor clearly invades and metastasizes to multiple organs, strengths of the model are its demonstration of invasive, angiogenic, colonization, and extravasation mechanisms found in most cancers. The efficient, convenient, and cost-effective method in which agents can be screened is also a distinct advantage. The most compelling limitation of the model system may be its reliance on a cell line to establish the primary tumor. Although the molecular characteristics of both breast and lung cancer cell lines have been shown to closely match their original human tumor [29, 30], the capacity of a cell line to maintain clonal heterogeneity has been questioned [31, 32]. While the NCI-H460 cell line used in these studies does exhibit invasive and metastatic properties, other important characteristics, such as drug resistance, may be lost or muted through serial passaging. Much like the National Cancer Institute in vitro drug discovery program [1], multiple models representing all of the lung cancer histologies would best test the potential clinical value of a new anticancer agent. However, in pilot studies, we have found that not all cell lines are as useful as the NCI-H460 for producing a model that rapidly metastasizes to multiple sites. Implantation of orthotopic tumor fragments derived from the A549 and NCI-H125 lung cancer cell lines resulted in slower and less extensive metastasis (unpublished data). Better yet, a model system that incorporated fresh human tumor to establish the model might ensure maintenance of human tumor heterogeneity more closely. To document reproducibility so that comparisons between drugs could be made, these human tumors would need to be passaged serially in generations of animals without creating cell lines. Our laboratory is in the process of establishing this type of model system.

In conclusion, this orthotopic lung cancer model system is capable of detecting drug activity in vivo against both primary and metastatic tumors using multiple end points. The model also conveys evidence of drug toxicity, and results may suggest certain possible mechanisms of action. The complex interactions between drug, tumor, and organism observed and assessed in this system closely reflect the human disease and may more accurately predict clinical outcomes in patients with lung cancer than other available models. Complete validation of the model can be assured only when prospective new drug studies in the model accurately predict response of the drug in clinical trials.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
The National Cancer Institute of Canada supported this research with funds from the Canadian Cancer Society.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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