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Experimental Therapeutics, Preclinical Pharmacology |
Institut de Recherches Servier, Division de Cancérologie Expérimentale, 92150 Suresnes, France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In each of the two models, all animals developed lung tumors, resulting
in 100% mortality. Histopathological study showed that these two
tumors spread locally to contiguous structures, including the
mediastinal pleura and diaphragm, with histological characteristics
consistent with the human pathology. Anticancer drugs used for the
treatment of NSCLC, such as cisplatin, doxorubicin, vinblastine,
and etoposide, enhanced the life span of treated mice in the two models
and were more active in the NCI-H460 than in the A549 model. The
increases of survival time as compared to control groups were from 60
(P 
0.05) to 83% (P 0.01)
and from 21 to 40% for NCI-H460 and A549, respectively. Vinorelbine,
paclitaxel, and irinotecan showed similar activities in the two models
and increased the survival of treated mice by between 38 and 79%
(P 0.001) and between 58 (P 0.01) and 78% in the NCI-H460 and A549 models, respectively.
However, none of these drugs was curative, reflecting the resistance of
this disease to chemotherapy.
S 16020-2 exhibited a remarkable antitumor activity, increasing the
survival by 82% (P 0.01) for NCI-H460 and by
126% (P 0.001) for A549. This drug was among
the most active compounds in these models, thereby indicating
its potential for the chemotherapy of this disease.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although s.c. xenograft models have been widely used to evaluate the antitumor activity of new compounds, they present major disadvantages in that tumor cells do not metastasize, and the parameter of animal survival cannot be used. The implantation of the tumor in the organ specific orthotopic site leads to an increased tumorigenicity and metastatic potential as compared to the ectopic s.c. models and thus could be more relevant to the clinical situation (3 , 4) .
Lung orthotopic models have been developed using intrabronchial instillation, intrathoracic or i.v. graft of tumor cell suspensions (5, 6, 7) , and implantation of histologically intact tumor tissue directly after surgery or biopsy (8) . A comparison of orthotopic and s.c. models showed that NSCLC tumors implanted intrathoracically into nude mice were almost always fatal (92%), in contrast to those implanted s.c. (3) .
The large cell carcinoma NCI-H460 and the adenocarcinoma A549 cell lines are tumorigenic in immunosuppressed mice when implanted s.c. (9) , and lung tumors are obtained after i.v. injection of A549 tumor cells (10) . These two cell lines have also been shown to be tumorigenic when injected via intrathoracic route into the pleural space of nude mice, producing, in the case of A549, metastases in the mediastinum (11) . However, these models have not been fully characterized with respect to tumor invasion, distant metastases, and sensitivity to anticancer drugs. To study the relevance of tumor orthotopic models to the clinical setting, we implanted each of these two NSCLC cell lines into the pleural cavity of nude mice. The histological characteristics and the growth and dissemination patterns of each tumor were analyzed. Their chemosensitivity was investigated using drugs currently used in this pathology, as well as promising new chemotherapeutic agents.
The antitumor effect of S 16020-2, a new topoisomerase II inhibitor (12) currently in Phase II clinical evaluation, was also investigated. S 16020-2 has shown a broad range of antitumor activity in a panel of murine and human tumor models (13) and was particularly active against tumors of pulmonary origin implanted i.v. or s.c. (9 , 10) .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice and Tumor Models.
Female athymic BALB/c nude mice were obtained from Iffa Credo (Lyon,
France) and weighed 20–22 g at the start of the experiments. The mice
were housed in sterilized filter-topped cages and maintained in sterile
conditions.
The human lung tumor cell lines NCI-H460 and A549 were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 (Life Technologies, Inc., Cergy Pontoise, France) complemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES buffer, pH 7.4. Cells were maintained at 37°C in 5% CO2/95% air. On the day of implantation (day 0), cells were harvested by incubation with trypsin, washed, and diluted in culture medium. Cell viability was determined by trypan blue dye exclusion and was greater than 95%. Animals were anesthetized with 2% Rompun (Bayer Pharma, Puteaux, France) at 5 mg/kg and Zoletil 100 (VirbacR, Carros, France) at 30 mg/kg, administered i.p. Tumor cells (106 cells) were implanted through the chest wall into the left pleural space of nude mice (i.pl.) in a volume of 100 µl using a 26 gauge needle. The depth of needle penetration through the intercostal muscles was controlled to avoid lung injury and hemorrhage into the pleural space. Prior to being returned to their cages, mice were placed until recovery under a heat lamp to maintain body temperature.
Histological Study.
The growth pattern of each tumor was first characterized. For this
purpose, 18 nude mice were inoculated i.pl. with
106 cells, and a subset of tumor-bearing animals
was sacrificed and autopsied on the indicated days. Organs were removed
and fixed in 10% phosphate-buffered formalin and embedded in paraffin.
Sections of 4 µm were stained with H&E for microscopic evaluation and
examined by a pathologist.
In Vivo Antitumor Activity.
All agents were administered i.v. at two or three doses. Doses
causing early death were considered to be toxic. The weight loss of
treated animals could not be used as a criterion of drug-induced
toxicity because it is principally related to disease progression. The
treatments were initiated when the tumor has begun to invade the
surrounding tissues, as shown by the histological study, 7 and 14 days
after the injection of NCI-H460 and A549 tumor cells, respectively. In
most cases, drugs were administered once a week, on days 7 and 14, or
on days 14, 21 and 28 to NCI-H460- and A549-bearing mice, respectively.
Each treated group consisted of 5–7 mice, and control groups consisted
of 6–14 mice. Animal mortality was checked daily, and the antitumor
activity was evaluated as follows: T/C % = MST of treated group/MST of
control group x 100. Results were also expressed as the
percentage of ILS (T/C of treated group - 100). The optimal dose
was the dose giving the highest T/C without toxic death.
Statistical Method.
A comparison of the survival curves between all of the treated and
control groups was performed with a log-rank test, which takes censored
values into account. If the log-rank 
2 was
significant (P 0.05), the comparison of each treated
group to the control group was done with a log-rank test followed by a
Holm’s adjustment to control the overall risk at 5%.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NCI-H460.
At an early stage of the disease, a local mediastinal invasion was
observed in 50% of mice on day 5 and in 100% on day 7 (Table 1)
and was confirmed by histological
analysis. Numerous tumor nodules of 1–5 mm in diameter were detected
on the diaphragm 5 days after the graft of the cells. The tumor
invasion of the mediastinal space preceded the presence of tumor
nodules in the lungs on day 7 (Table 1)
. Microinvasion of the lung
parenchyma by NCI-H460 tumor cells was observed on day 11 (Fig. 1A).
Five days after the
injection of tumor cells, the presence of pericardial nodules was
observed in 50% of mice, and on day 7, tumor cells began to invade the
peritoneum (not shown). At a more advanced stage of the disease (days
14–25), tumor cells had totally invaded the thoracic cavity, and
distant metastatic sites were seen in peritoneal organs, such as the
liver, stomach, and pancreas (Fig. 1B
and Table 1
). Tumor
nodules around the mesenteric lymph nodes were also observed on days 19
and 25 (Fig. 1C).
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. A
massive invasion of the mediastinal space by tumor cells, and
particularly the posterior mediastinal space around the esophagus, was
observed on days 11 and 14 (Fig. 1D).
A549 cells were
organized in tubular structures characteristic of an adenocarcinoma. At
this stage, pericardial nodules of tumor cells were observed in 50% of
mice. On day 14, tumor nodules were detected on the lungs (Table 1)
,
and malignant cells had begun to invade the lung parenchyma. The
invasion of the diaphragm (Fig. 1E)
preceded the
dissemination of the tumor cells in the peritoneum. Pleural effusion in
thoracic cavity was observed three weeks after the injection of tumor
cells, indicating an advanced disease. The presence of tumor nodules
was systematically observed on the peritoneum membrane, but there was
no dissemination to the organs. Abundant ascites were observed from
days 21 to 39 (not shown). At a more advanced stage (day 31),
perivertebral tumor nodules were detected (Fig. 1F).
Chemotherapy Experiments.
To study the chemosensitivity of these two models, clinically used
anticancer drugs (cisplatin, doxorubicin, etoposide, cyclophosphamide,
and vinblastine) and new drugs under investigation (paclitaxel,
vinorelbine, gemcitabine, irinotecan, and topotecan) were administered
to tumor-bearing mice. To better mimic the clinical situation,
treatment began only when the disease was developed, 7 and 14 days
after the injection of NCI-H460 and A549 tumor cells, respectively. All
of the compounds were tested at least twice in separate experiments,
giving similar results. Table 2
shows the
results of a representative experiment for each drug.
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0.05). This compound was less active
against A549, the maximum ILS being 35% at 8 mg/kg.
Vinblastine was significantly active against NCI-H460 tumor when
administered at 1–4 mg/kg with T/C values ranging from 137 to
183% (P 0.01). This compound showed antitumor
activity against the A549 tumor model when administered at 2 mg/kg
(T/C = 137%).
Etoposide administered at 35 and 70 mg/kg induced an ILS of NCI-H460-bearing mice of 66 and 76%, respectively. In the A549 model, etoposide showed a marginal activity, with a maximum T/C of 121% when administered at the highest dosage.
Doxorubicin, administered at 5 and 10 mg/kg to NCI-H460-bearing mice,
showed a statistically significant antitumor activity with T/C values
of 151 (P 0.001) and 160% (P
0.05), respectively. This compound was less active against the A549
tumor, inducing a maximum ILS of 40%.
Cyclophosphamide was inactive against the NCI-H460 tumor and showed a marginal activity against A549 tumor when administered at 200 mg/kg (T/C = 129%).
Gemcitabine was active only against the NCI-H460 tumor with a T/C value
of 138% when administered at 400 mg/kg. Paclitaxel showed antitumor
activity in these two models when administered at 20 and 40 mg/kg. This
activity was significant in the A549 tumor, with a maximum T/C of 158%
(P 0.01).
Vinorelbine, which was administered at 5 and 10 mg/kg to NCI-H460
tumor-bearing mice, showed a significant antitumor activity, with T/C
values of 146 and 179% (P 0.001), respectively.
Administered at 2.5–10 mg/kg to A459-bearing mice, its antitumor
activity was dose dependent and statistically significant at 10 mg/kg
(T/C = 174%; P 0.01).
Irinotecan, administered at 10–40 mg/kg, was found to be active in the two models. The maximum T/C values were 161% at 40 mg/kg in NCI-H460 and 178% at 20 mg/kg in A549. In contrast, topotecan was only active in the NCI-H460 tumor model when administered at 1.25 and 2.5 mg/kg (T/C = 159 and 164%, respectively). Administered at 5 mg/kg, this compound was found to be toxic in the two models because early deaths were observed.
The antitumor activity of S 16020-2 was also investigated. It was
administered at 20, 40, and 80 mg/kg, the latter dose being the maximum
tolerated dose in nude mice. The activity of S 16020-2 was dose
dependent and maximum at 80 mg/kg, with T/C values of 182
(P 0.01) and 226% (P 0.001) for
NCI-H460 and A549, respectively (Table 2)
. Fig. 2
shows the effect of these three doses
of S 16020-2 on the survival of animals bearing the A549 tumor. The ILS
of treated mice was statistically significant after administration of
40 (P 0.01) and 80 mg/kg (P
0.001). To study the effect of S 16020-2 on the pattern of tumor
dissemination, all of the mice were sacrificed just before the
anticipated death of control mice. Macroscopic examination (not shown)
showed both a reduced extent of tumor dissemination and a lack of
pleural effusion in mice treated by S 16020-2, indicating a delay in
the development of the disease.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The NCI-H460 and A549 human NSCLCs were implanted in the pleural cavity of nude mice to obtain orthotopic tumor models that could be closer to the clinical situation and could be used to identify new compounds active against this pathology. After tumor cell implantation, 100% of the animals died, with a MST ranging from 19.5 to 27.5 days and from 36.5 to 42.0 days for NCI-H460 and A549, respectively.
Our study shows that NCI-H460 and A549 tumor cells grow in the pleural cavity and invade contiguous structures, including diaphragm, mediastinum, and lung parenchyma. In addition, they spread to distant sites in the peritoneum, as observed in the human disease, and induced clinical symptoms of cachexia and dypsnea (15) . More interestingly, pericardial tumor nodules were detected in half of the mice, as is frequently observed in the human pathology (15) . At a more advanced stage of the disease, a pleural effusion was found in mice bearing A549 cells but not NCI-H460 cells. In the human disease, a pleural effusion develops in a majority of patients having primary lung cancer (16) and lung adenocarcinoma (17) .
Taken together, these data show that the growth patterns of NSCLC cells implanted intrapleurally were similar to that encountered in lung cancer patients. Moreover, these two orthotopic models mimic, in a few weeks, an advanced stage of the human disease.
The overall outcome of advanced NSCLC remains poor. In the case of metastatic disease, chemotherapy has been widely used for the management of inoperable adenocarcinoma (1 , 18) . Prior to 1990, some drugs, such as cisplatin, mitomycin-c, ifosfamide, vindesine, vinblastine, doxorubicin, and etoposide, were shown to have significant antitumor activity when used as single agent, but responses were only partial and of short duration (19) . In the past few years, new active drugs, such as vinorelbine, paclitaxel, docetaxel, gemcitabine, and, more recently, irinotecan and topotecan, were found to improve survival and relieve symptoms in advanced stage patients (20 , 21) . Most of these drugs were tested in the two models to determine their sensitivity and their predictivity.
All of the drugs tested demonstrated some antitumor activity except for cyclophosphamide and gemcitabine in the NCI-H460 and A549 tumor model, respectively. Cisplatin, vinblastine, etoposide, and doxorubicin were more active in the NCI-H460 than in the A549 model, showing that the A549 adenocarcinoma seemed to be more resistant to the chemotherapeutic agents than the NCI-H460 carcinoma. The most active compound in the two models was vinorelbine, which was approved by the Food and Drug Administration for NSCLC and induced objective response rates of at least 20% in randomized studies (22, 23, 24) .
We also investigated the antitumor activity of paclitaxel, which has shown clinical activity in a number of tumor types, including ovarian adenocarcinoma and metastatic breast cancer (25) . In chemotherapy for NSCLC, response rate of paclitaxel used as a single agent is about 20–25% (26 , 27) . Paclitaxel was more active against A549 than against NCI-H460 tumors.
Irinotecan and topotecan, which belong to the family of camptothecins, have recently entered clinical trials against lung cancer. These compounds were administered following a repeated schedule, according to the published pharmacokinetic data (28 , 29) . Irinotecan demonstrated antitumor activity in both models, whereas topotecan was active only in the NCI-H460 model. In the clinic, topotecan has been shown to have only a limited activity in the treatment of NSCLC (21) , although irinotecan is significantly active, with an objective response rate of 27% (30 , 31) .
Together, these data show that most of the drugs reported to be clinically effective were also active in our models. The fact that none of these drugs used as a single agent were found to be curative in these models, which mimic an advanced stage of lung carcinoma, is consistent with the poor response of this disease to monochemotherapy (21) .
We also report the significant antitumor activity of a new olivacine derivative, S 16020-2. In the A549 model, S 16020-2 treatment increased more than 2-fold the life span of treated mice. At an advanced stage of the disease, no pleural effusion was observed in mice treated by S 16020-2, indicating that treatment with S 16020-2 delayed the development of the disease. Again, as for the other drugs, S 16020-2 did not induce long-term survivors. Among the antitumor agents tested, S 16020-2 was the most active compound in the A549 model and was at least as active as vinorelbine, vinblastine, and etoposide in the NCI-H460 model. The fact that S 16020-2 was at least as active as vinorelbine in the two models is an encouraging result, with regard to the proven clinical activity of vinorelbine in this pathology (20 , 22 , 23) .
Differences in sensitivity to various chemotherapeutic agents of experimental tumors growing in orthotopic or s.c. sites have been reported, and the organ microenvironment has been shown to influence the response of metastases to chemotherapy in experimental animals (32) . We have previously shown that S 16020-2 was found to be active when A549 and NCI-H460 tumors were implanted s.c. (9) . In contrast, doxorubicin, found to be active in the present study in the case of NCI-H460 implanted i.pl., showed only a marginal activity when this tumor was implanted s.c. (9) . These results thus corroborate observations that the response to antitumor agents may be dependent on the site of the tumor implantation. Hence, the use of new orthotopic models of NSCLC could be helpful in the search of new, more active therapeutic agents in this pathology.
Finally, the antitumor activity of S 16020-2 against two highly metastatic models of NSCLC delineates an interesting chemotherapeutic potential for this drug in this disease alone and in combination with other active chemotherapeutic agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Institut de Recherches Servier, Division de
Cancérologie Expérimentale, 11 rue des Moulineaux,
92150 Suresnes, France. Phone: 33-1-55-72-24-10; Fax:
33-1-55-72-24-40. 
2 The abbreviations used are: ILS, increase in
life span; i.pl., intrapleural; MST, median survival time; NSCLC,
non-small cell lung carcinoma; T/C, treated versus
control.
Received 7/ 9/99; revised 10/ 6/99; accepted 10/ 7/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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