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Histology and Sensitivity to Anticancer Drugs of Two HumanNon-Small Cell Lung Carcinomas Implanted in the PleuralCavity of Nude Mice

Laurence Kraus-Berthier,1 Michel Jan,Nicolas Guilbaud, Monique Naze, Alain Pierre,and Ghanem AtassiInstitut de Recherches Servier, Division de Cance´rologieExperimentale, 92150 Suresnes, France

ABSTRACTWe have established two metastatic models of human

non-small cell lung carcinoma (NSCLC)—the NCI-H460large-cell carcinoma and the A549 adenocarcinoma—by in-oculating tumor cells into the pleural space of nude mice.The objectives of this work were as follows: (a) to study thehistological characteristics and growth and disseminationpatterns of these tumors in nude mice; (b) to assess theirsensitivity to drugs that have demonstrated significant clin-ical therapeutic effect in the treatment of NSCLC; and (c) toinvestigate the antitumor activity of S 16020-2, a new oli-vacine derivative, currently in Phase II clinical evaluation.

In each of the two models, all animals developed lungtumors, resulting in 100% mortality. Histopathologicalstudy showed that these two tumors spread locally to con-tiguous structures, including the mediastinal pleura anddiaphragm, with histological characteristics consistent withthe human pathology. Anticancer drugs used for the treat-ment of NSCLC, such as cisplatin, doxorubicin, vinblastine,and etoposide, enhanced the life span of treated mice in thetwo models and were more active in the NCI-H460 than inthe A549 model. The increases of survival time as comparedto 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 activ-ities in the two models and increased the survival of treatedmice 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 andby 126% (P < 0.001) for A549. This drug was among the

most active compounds in these models, thereby indicatingits potential for the chemotherapy of this disease.

INTRODUCTIONLung cancer is one of the leading causes of cancer-related

deaths in adults and continues to show an increasing incidence.NSCLC2 is the most common type of lung cancer, accountingfor about 80% of lung cancers. The diagnosis is frequently madein patients with advanced-stage disease, and the overall responserate to available chemotherapy was lower than 19% before 1990(1). In the past few years, the new chemotherapeutic agentsvinorelbine, paclitaxel, docetaxel, and gemcitabine have dem-onstrated significant antitumor activity in the treatment of ad-vanced NSCLC, producing objective response rates of at least20% (2). The severity of the prognosis of advanced broncho-pulmonary carcinomas, however, justifies the development ofnovel and potentially more active drugs.

Although s.c. xenograft models have been widely used toevaluate the antitumor activity of new compounds, they presentmajor disadvantages in that tumor cells do not metastasize, andthe parameter of animal survival cannot be used. The implan-tation of the tumor in the organ specific orthotopic site leads toan increased tumorigenicity and metastatic potential as com-pared to the ectopic s.c. models and thus could be more relevantto the clinical situation (3, 4).

Lung orthotopic models have been developed using intra-bronchial instillation, intrathoracic or i.v. graft of tumor cellsuspensions (5–7), and implantation of histologically intact tu-mor tissue directly after surgery or biopsy (8). A comparison oforthotopic and s.c. models showed that NSCLC tumors im-planted intrathoracically into nude mice were almost alwaysfatal (92%), in contrast to those implanted s.c. (3).

The large cell carcinoma NCI-H460 and the adenocarci-noma A549 cell lines are tumorigenic in immunosuppressedmice when implanted s.c. (9), and lung tumors are obtained afteri.v. injection of A549 tumor cells (10). These two cell lines havealso been shown to be tumorigenic when injected via intratho-racic route into the pleural space of nude mice, producing, in thecase of A549, metastases in the mediastinum (11). However,these models have not been fully characterized with respect totumor invasion, distant metastases, and sensitivity to anticancerdrugs. To study the relevance of tumor orthotopic models to theclinical setting, we implanted each of these two NSCLC celllines into the pleural cavity of nude mice. The histologicalcharacteristics and the growth and dissemination patterns of

Received 7/9/99; revised 10/6/99; accepted 10/7/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 To whom requests for reprints should be addressed, at Institut deRecherches Servier, Division de Cance´rologie Experimentale, 11 ruedes 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., intrapleu-ral; MST, median survival time; NSCLC, non-small cell lung carci-noma; T/C, treatedversuscontrol.

297Vol. 6, 297–304, January 2000 Clinical Cancer Research

each tumor were analyzed. Their chemosensitivity was investi-gated using drugs currently used in this pathology, as well aspromising new chemotherapeutic agents.

The antitumor effect of S 16020-2, a new topoisomerase IIinhibitor (12) currently in Phase II clinical evaluation, was alsoinvestigated. S 16020-2 has shown a broad range of antitumoractivity in a panel of murine and human tumor models (13) andwas particularly active against tumors of pulmonary originimplanted i.v. or s.c. (9, 10).

MATERIALS AND METHODSDrugs. Cisplatin (Cisplatine), gemcitabine (Gemzar),

and vinblastine (Velbe) were supplied by Lilly (Saint-Cloud,France); cyclophosphamide (Endoxan) was supplied by Sarget(Merignac, France); doxorubicin (Adriblastine) was supplied byFarmitalia Carlo Erba (Pharmacia & Upjohn, St Quentin-en-Yvelines, France); irinotecan (Campto) was supplied by Bellon(Rhone-Poulenc Rorer, Montrouge, France); topotecan (Hycam-tin) was supplied by Smithkline Beecham (Nanterre, France);and vinorelbine (Navelbine) was supplied by Pierre Fabre On-cology (Boulogne, France). S 16020-2 was synthesized in ourinstitute as described (14). These drugs were solubilized anddiluted in sterile water. Paclitaxel (Taxol®, Sigma) was dis-solved at 40 mg/ml in a mixture of 50% ethanol and 50%chremophor (v/v) and diluted in water to the desired concentra-tion. Etoposide (Ve´peside, Sigma) was dissolved in DMSO(3.5% final volume) and diluted in saline solution containing6.5% Tween 80 to the desired concentration. All drugs wereadministered to animals at 0.1 ml/10 g of body weight by i.v.route in the caudal tail vein on the indicated days.

Mice and Tumor Models. Female athymic BALB/cnude mice were obtained from Iffa Credo (Lyon, France) andweighed 20–22 g at the start of the experiments. The mice werehoused in sterilized filter-topped cages and maintained in sterileconditions.

The human lung tumor cell lines NCI-H460 and A549 wereobtained from the American Type Culture Collection (Manas-sas, 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/mlpenicillin, 100mg/ml streptomycin, and 10 mM HEPES buffer,pH 7.4. Cells were maintained at 37°C in 5% CO2/95% air. Onthe day of implantation (day 0), cells were harvested by incu-bation with trypsin, washed, and diluted in culture medium. Cellviability was determined by trypan blue dye exclusion and wasgreater 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. Tumorcells (106 cells) were implanted through the chest wall into theleft pleural space of nude mice (i.pl.) in a volume of 100mlusing a 26 gauge needle. The depth of needle penetrationthrough the intercostal muscles was controlled to avoid lunginjury and hemorrhage into the pleural space. Prior to beingreturned to their cages, mice were placed until recovery under aheat lamp to maintain body temperature.

Histological Study. The growth pattern of each tumorwas first characterized. For this purpose, 18 nude mice wereinoculated 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-bufferedformalin and embedded in paraffin. Sections of 4mm werestained with H&E for microscopic evaluation and examined bya pathologist.

In Vivo Antitumor Activity. All agents were adminis-tered i.v. at two or three doses. Doses causing early death wereconsidered to be toxic. The weight loss of treated animals couldnot be used as a criterion of drug-induced toxicity because it isprincipally related to disease progression. The treatments wereinitiated when the tumor has begun to invade the surroundingtissues, as shown by the histological study, 7 and 14 days afterthe injection of NCI-H460 and A549 tumor cells, respectively.In most cases, drugs were administered once a week, on days 7and 14, or on days 14, 21 and 28 to NCI-H460- and A549-bearing mice, respectively. Each treated group consisted of 5–7mice, and control groups consisted of 6–14 mice. Animal mor-tality was checked daily, and the antitumor activity was evalu-ated as follows: T/C %5 MST of treated group/MST of controlgroup3 100. Results were also expressed as the percentage ofILS (T/C of treated group2 100). The optimal dose was thedose giving the highest T/C without toxic death.

Statistical Method. A comparison of the survival curvesbetween all of the treated and control groups was performedwith a log-rank test, which takes censored values into account.If the log-rankx2 was significant (P # 0.05), the comparison ofeach treated group to the control group was done with a log-ranktest followed by a Holm’s adjustment to control the overall riskat 5%.

RESULTSHistological Study. After inoculation of 106 tumor cells

into the pleural space, nude mice progressively became dyspneicand cachectic, and death occurred in 100% of the animals witha MST of 22.4 days for NCI-H460 and 38.8 days for A549.Mice were sacrificed and autopsied at the indicated days, and anhistological study was performed to appreciate the dissemina-tion and the progression of the disease.

NCI-H460. At an early stage of the disease, a localmediastinal invasion was observed in 50% of mice on day 5 andin 100% on day 7 (Table 1) and was confirmed by histologicalanalysis. Numerous tumor nodules of 1–5 mm in diameter weredetected on the diaphragm 5 days after the graft of the cells. Thetumor invasion of the mediastinal space preceded the presenceof tumor nodules in the lungs on day 7 (Table 1). Microinvasionof the lung parenchyma by NCI-H460 tumor cells was observedon day 11 (Fig. 1A). Five days after the injection of tumor cells,the presence of pericardial nodules was observed in 50% ofmice, and on day 7, tumor cells began to invade the peritoneum(not shown). At a more advanced stage of the disease (days14–25), tumor cells had totally invaded the thoracic cavity, anddistant metastatic sites were seen in peritoneal organs, such asthe liver, stomach, and pancreas (Fig. 1B and Table 1). Tumornodules around the mesenteric lymph nodes were also observedon days 19 and 25 (Fig. 1C).

A459. On day 4, the primary tumor had already spreadlocally to continuous structures, including mediastinum, lung,and diaphragm (Table 1). A massive invasion of the mediastinal

298 Chemosensitivity of Two Models of Human NSCLC

space by tumor cells, and particularly the posterior mediastinalspace around the esophagus, was observed on days 11 and 14(Fig. 1D). A549 cells were organized in tubular structurescharacteristic of an adenocarcinoma. At this stage, pericardialnodules of tumor cells were observed in 50% of mice. On day14, tumor nodules were detected on the lungs (Table 1), andmalignant cells had begun to invade the lung parenchyma. Theinvasion of the diaphragm (Fig. 1E) preceded the disseminationof the tumor cells in the peritoneum. Pleural effusion in thoraciccavity was observed three weeks after the injection of tumorcells, indicating an advanced disease. The presence of tumornodules was systematically observed on the peritoneum mem-brane, but there was no dissemination to the organs. Abundantascites were observed from days 21 to 39 (not shown). At amore advanced stage (day 31), perivertebral tumor nodules weredetected (Fig. 1F).

Chemotherapy Experiments. To study the chemosensi-tivity of these two models, clinically used anticancer drugs(cisplatin, doxorubicin, etoposide, cyclophosphamide, and vin-blastine) and new drugs under investigation (paclitaxel, vinorel-bine, gemcitabine, irinotecan, and topotecan) were administeredto tumor-bearing mice. To better mimic the clinical situation,treatment began only when the disease was developed, 7 and 14days after the injection of NCI-H460 and A549 tumor cells,respectively. All of the compounds were tested at least twice inseparate experiments, giving similar results. Table 2 shows theresults of a representative experiment for each drug.

Cisplatin showed a dose-dependent activity in the NCI-H460 tumor model when administered at 2–8 mg/kg. The in-creases in life span were 33 and 62% after the administration of4 and 8 mg/kg, respectively, and there was statistically signifi-cant difference from the control at 8 mg/kg (P # 0.05). Thiscompound was less active against A549, the maximum ILSbeing 35% at 8 mg/kg.

Vinblastine was significantly active against NCI-H460 tu-mor when administered at 1–4 mg/kg with T/C values rangingfrom 137 to 183% (P # 0.01). This compound showed antitu-mor activity against the A549 tumor model when administeredat 2 mg/kg (T/C5 137%).

Etoposide administered at 35 and 70 mg/kg induced an ILSof NCI-H460-bearing mice of 66 and 76%, respectively. In theA549 model, etoposide showed a marginal activity, with amaximum T/C of 121% when administered at the highestdosage.

Doxorubicin, administered at 5 and 10 mg/kg to NCI-H460-bearing mice, showed a statistically significant antitumoractivity with T/C values of 151 (P # 0.001) and 160% (P #0.05), respectively. This compound was less active against theA549 tumor, inducing a maximum ILS of 40%.

Cyclophosphamide was inactive against the NCI-H460 tu-mor and showed a marginal activity against A549 tumor whenadministered at 200 mg/kg (T/C5 129%).

Gemcitabine was active only against the NCI-H460 tumorwith a T/C value of 138% when administered at 400 mg/kg.Paclitaxel showed antitumor activity in these two models whenadministered at 20 and 40 mg/kg. This activity was significantin the A549 tumor, with a maximum T/C of 158% (P # 0.01).

Vinorelbine, which was administered at 5 and 10 mg/kg toNCI-H460 tumor-bearing mice, showed a significant antitumoractivity, with T/C values of 146 and 179% (P # 0.001), respec-tively. Administered at 2.5–10 mg/kg to A459-bearing mice, itsantitumor activity was dose dependent and statistically signifi-cant at 10 mg/kg (T/C5 174%;P # 0.01).

Irinotecan, administered at 10–40 mg/kg, was found to beactive 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. Incontrast, topotecan was only active in the NCI-H460 tumormodel when administered at 1.25 and 2.5 mg/kg (T/C5 159 and164%, respectively). Administered at 5 mg/kg, this compoundwas found to be toxic in the two models because early deathswere observed.

The antitumor activity of S 16020-2 was also investi-gated. It was administered at 20, 40, and 80 mg/kg, the latterdose being the maximum tolerated dose in nude mice. Theactivity of S 16020-2 was dose dependent and maximum at80 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

Table 1 Histological analysis of tumor dissemination in the intrapleural lung cancer models

Day Heart Lung Mediastinum Diaphragm Liver Duodenum Stomach PancreasMesenteric

lymph nodes

NCI-H4605 1/2 N 1/2 1 N N N N N7 1/2 11 1 11 N N N N N

11 N 111 11 11 N N N N N14 1/2 111 111 111 1/2 N 1/2 1/2 N19 1/2 111 111 111 11 N 1/2 1/2 1/225 1/2 111 111 111 11 1/2 11 111 11

A5494 N 1/2 1/2 1 N N N N N

11 1/2 1 11 1 N N N N N14 1/2 11 11 11 N N N N N21 1/2 11 11 11 1/2 N N N N31 1/2 11 111 111 N N N N N39 N 11 111 11 N N N N N

Tumor cells (106) were implanted in the pleural space of nude mice on day 0. Two or three mice were sacrificed on each of the indicated days,and organs were removed and fixed for histopathological characterization.1/2, at least one mouse presenting tumor nodules;1 to 111, all micepresenting tumor nodules, from a few nodules (1) to a massive invasion (111). N, normal.

299Clinical Cancer Research

survival of animals bearing the A549 tumor. The ILS oftreated mice was statistically significant after administrationof 40 (P # 0.01) and 80 mg/kg (P # 0.001). To study theeffect of S 16020-2 on the pattern of tumor dissemination, allof the mice were sacrificed just before the anticipated deathof 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, indi-cating a delay in the development of the disease.

DISCUSSIONThe organ-specific environment is an important factor for

the growth and progression of tumorsin vivo (4). In the case of

Fig. 1 Growth and dissemination of the NCI-H460 and A549 tumors after intrapleural implantation.A,day 11: microinvasion of the pulmonary tissueby NCI-H460 tumor cells. Tumor nodules were seen in the lung (arrow). 3 100.B, day 14: presence of NCI-H460 tumor nodules in the gastric serosa(arrow). 3 25. C, day 25: presence of NCI-H460 tumor nodules around the mesenteric node (arrow). 3 100. D, day 14: massive invasion of theposterior mediastinal space and tubular proliferation of A549 tumor cells around the esophagus (arrow). 3 25. E, day 21: invasion and infiltrationof the diaphragm by A549 tumor cells (arrow). 3 200.F, day 31: perivertebral tumor nodules of A549 tumor cells (arrow). 3 25.


NSCLC, it was previously reported that intrabronchially im-planted lung tumors were not highly invasive and that theirgrowth was limited to the lung parenchyma and the right lung(5). When these tumor cells were implanted s.c or i.v., nointrapleural metastases were observed (6–8).

The NCI-H460 and A549 human NSCLCs were implantedin the pleural cavity of nude mice to obtain orthotopic tumormodels 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 aMST ranging from 19.5 to 27.5 days and from 36.5 to 42.0 daysfor NCI-H460 and A549, respectively.

Our study shows that NCI-H460 and A549 tumor cellsgrow in the pleural cavity and invade contiguous structures,including diaphragm, mediastinum, and lung parenchyma. Inaddition, they spread to distant sites in the peritoneum, as

Table 2 Activity of anticancer drugs in the NCI-H460 and A549 orthotopic tumor modelsThe survival curve of each treated group was compared with that of control by the log-rank test.

DrugDose

(mg/kg/day)

NCI-H460 A549

Schedule MST (range), days T/C (%) Schedule MST (range), days T/C (%)

Cisplatin 2 Days 7, 14 36.0 (23–30) 108 NTa

4 Days 7, 14 32.0 (21–37) 133 Days 14, 21, 28 48.3 (35–57) 1238 Days 7, 14 39.0 (35–42) 162b Days 14, 21, 28 53.0 (44–55) 135

Control 24.1 (19–42) 100 39.3 (33–43) 100

Vinblastine 1 Days 7, 14 27.8 (27–31) 137c NT2 Days 7, 14 32.0 (26–41) 158c Days 14, 21, 28 44.3 (41–69) 1134 Days 7, 14 37.0 (29–44) 183c Days 14, 21, 28 54.0 (31–55) 137

Control 20.3 (19–27) 100 39.3 (33–43) 100

Etoposide 17.5 Days 7, 10, 14 24.7 (21–25) 109 NT35 Days 7, 10, 14 37.7 (33–38) 166 Days 14, 17, 21 43.0 (37–49) 11570 Days 7, 10, 14 40.0 (10–44) 176 Days 14, 17, 21 45.0 (37–76) 121

Control 22.7 (19–35) 100 37.3 (28–63) 100

Cyclophosphamide 50 Days 7, 14 21.7 (19–22) 100 Days 14, 21, 28 36.5 (29–38) 87100 Days 7, 14 23.8 (18–29) 109 Days 14, 21, 28 42.5 (39–45) 101200 Days 7, 14 23.3 (15–24) 107 Days 14, 21, 28 54.0 (29–56) 129

Control 21.8 (19–27) 100 42.0 (24–88) 100

Doxorubicin 5 Days 7, 14 30.5 (25–65) 151d Days 14, 21, 28 48.0 (37–65) 122e

10 Days 7, 14 32.5 (18–45) 160b Days 14, 21, 28 55.3 (46–66) 140Control 20.3 (19–23) 100 39.3 (33–43) 100

Paclitaxel 10 Days 7, 14 NT Days 14, 21, 28 47.3 (29–48) 10020 Days 7, 14 38.0 (28–45) 138 Days 14, 21, 28 56.8 (47–57) 146b

40 Days 7, 14 35.0 (31–37) 127 Days 14, 21, 28 61.3 (57–70) 158c

Control 27.5 (22–46) 100 38.8 (27–50) 100

Vinorelbine 2.5 NT Days 14, 21, 28 41.3 (29–48) 1095 Days 7, 14 34.0 (18–48) 146d Days 14, 21, 28 52.3 (42–59) 138

10 Days 7, 14 41.8 (35–80) 179d Days 14, 21, 28 66.0 (54–83) 174c

Control 23.3 (19–24) 100 38.0 (33–68) 100

Gemcitabine 100 Days 7, 14 23.8 (22–37) 112 Days 14, 21, 28 38.0 (22–44) 95200 Days 7, 14 22.8 (20–25) 127 Days 14, 21, 28 37.0 (32–46) 93400 28.0 (19–30) 138 Days 14, 21, 28 41.0 (18–53) 103

Control 20.3 (19–23) 100 40.0 (27–69) 100

Irinotecan 10 Days 5–9 29.8 (27–31) 153 Days 14–19 37.8 (29–39) 10320 Days 5–9 29.7 (24–30) 152 Days 14–19 65.0 (36–96) 17840 Days 5–9 31.3 (29–32) 161 Days 14–19 49.8 (35–53) 136

Control 19.5 (16–37) 100 36.5 (28–57) 100

Topotecan 1.25 Days 5–9 31.0 (26–42) 159b Days 14–19 44.0 (40–76) 1062.5 Days 5–9 32.0 (31–33) 164 Days 14–19 47.0 (33–104) 1135 Days 5–9 12.7 (12–13) 65 Days 14–19 21.0 (20–49) 136

Control 19.5 (16–37) 100 41.5 (24–86) 100

S 16020-2 20 Days 7, 14 27.3 (25–32) 122 Days 14, 21, 28 53.0 (39–69) 14340 Days 7, 14 31.0 (20–34) 139 Days 14, 21, 28 63.3 (48–98) 170c

80 Days 7, 14 40.7 (33–41) 182c Days 14, 21, 28 84.0 (65–104) 226d

Control 22.4 (18–36) 100 37.1 (21–76) 100a NT, not tested.b P # 0.05.c P # 0.01.d P # 0.001.e In this experiment, doxorubicin was administered at 4 and 8 mg/kg.


observed in the human disease, and induced clinical symp-toms of cachexia and dypsnea (15). More interestingly, peri-cardial tumor nodules were detected in half of the mice, as isfrequently observed in the human pathology (15). At a moreadvanced stage of the disease, a pleural effusion was found inmice bearing A549 cells but not NCI-H460 cells. In thehuman disease, a pleural effusion develops in a majority ofpatients having primary lung cancer (16) and lung adenocar-cinoma (17).

Taken together, these data show that the growth patterns ofNSCLC cells implanted intrapleurally were similar to that en-countered in lung cancer patients. Moreover, these two ortho-topic models mimic, in a few weeks, an advanced stage of thehuman disease.

The overall outcome of advanced NSCLC remains poor. Inthe case of metastatic disease, chemotherapy has been widelyused 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 assingle agent, but responses were only partial and of short dura-tion (19). In the past few years, new active drugs, such asvinorelbine, paclitaxel, docetaxel, gemcitabine, and, more re-cently, irinotecan and topotecan, were found to improve survivaland relieve symptoms in advanced stage patients (20, 21). Mostof these drugs were tested in the two models to determine theirsensitivity and their predictivity.

All of the drugs tested demonstrated some antitumor ac-tivity except for cyclophosphamide and gemcitabine in theNCI-H460 and A549 tumor model, respectively. Cisplatin, vin-blastine, etoposide, and doxorubicin were more active in theNCI-H460 than in the A549 model, showing that the A549adenocarcinoma seemed to be more resistant to the chemother-apeutic agents than the NCI-H460 carcinoma. The most activecompound in the two models was vinorelbine, which was ap-

proved by the Food and Drug Administration for NSCLC andinduced objective response rates of at least 20% in randomizedstudies (22–24).

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 paclitaxelused as a single agent is about 20–25% (26, 27). Paclitaxel wasmore active against A549 than against NCI-H460 tumors.

Irinotecan and topotecan, which belong to the family ofcamptothecins, have recently entered clinical trials against lungcancer. These compounds were administered following a re-peated schedule, according to the published pharmacokineticdata (28, 29). Irinotecan demonstrated antitumor activity in bothmodels, whereas topotecan was active only in the NCI-H460model. In the clinic, topotecan has been shown to have only alimited activity in the treatment of NSCLC (21), although iri-notecan is significantly active, with an objective response rate of27% (30, 31).

Together, these data show that most of the drugs reportedto be clinically effective were also active in our models. The factthat none of these drugs used as a single agent were found to becurative in these models, which mimic an advanced stage oflung carcinoma, is consistent with the poor response of thisdisease to monochemotherapy (21).

We also report the significant antitumor activity of a newolivacine derivative, S 16020-2. In the A549 model, S 16020-2treatment increased more than 2-fold the life span of treatedmice. At an advanced stage of the disease, no pleural effusionwas observed in mice treated by S 16020-2, indicating thattreatment with S 16020-2 delayed the development of the dis-ease. Again, as for the other drugs, S 16020-2 did not inducelong-term survivors. Among the antitumor agents tested,S 16020-2 was the most active compound in the A549 modeland was at least as active as vinorelbine, vinblastine, and

Fig. 2 Antitumor activity of S 16020-2in the A549 orthotopic tumor model.Mice were treated with S 16020-2 i.v.at the indicated doses 14, 21, and 28days after the implantation of 106

A549 tumor cells into the pleuralspace of nude mice. The survival of S16020-2-treated and control animalswas compared using the log-rank test:**, P # 0.01; ***, P # 0.001.


etoposide in the NCI-H460 model. The fact that S 16020-2 wasat least as active as vinorelbine in the two models is an encour-aging result, with regard to the proven clinical activity of vi-norelbine in this pathology (20, 22, 23).

Differences in sensitivity to various chemotherapeuticagents of experimental tumors growing in orthotopic or s.c.sites have been reported, and the organ microenvironmenthas been shown to influence the response of metastases tochemotherapy in experimental animals (32). We have previ-ously shown that S 16020-2 was found to be active whenA549 and NCI-H460 tumors were implanted s.c. (9). Incontrast, doxorubicin, found to be active in the present studyin the case of NCI-H460 implanted i.pl., showed only amarginal activity when this tumor was implanted s.c. (9).These results thus corroborate observations that the responseto antitumor agents may be dependent on the site of the tumorimplantation. Hence, the use of new orthotopic models ofNSCLC could be helpful in the search of new, more activetherapeutic agents in this pathology.

Finally, the antitumor activity of S 16020-2 against twohighly metastatic models of NSCLC delineates an interestingchemotherapeutic potential for this drug in this disease aloneand in combination with other active chemotherapeutic agents.

ACKNOWLEDGMENTSWe are grateful to Dr. Jean-Franc¸ois Boivin for histological anal-

ysis, to Dr. Gordon Tucker and Michael Burbridge for critical reading ofthe manuscript, and to Fre´derique Bertin for secretarial assistance.

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histology and sensitivity to anticancer drugs of two human ... · the human pathology. anticancer...

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