[CANCER RESEARCH 56. 3743-3746. Augusl 15. 19961

Doxorubicin Encapsulated in Sterically Stabilized Liposomes Is Superior to FreeDrug or Drug-containing Conventional Liposomes at Suppressing Growthand Métastasesof Human Lung Tumor Xenografts1

Takashi Sakakibara, Fang-An Chen, Hisashi Kida, Katsuyuki Kunieda, Rosa E. Cuenca, F. J. Martin,2 andRichard B. Bankert3

Department of Molecular Immunologi', Roswell Park Cancer Institute, Buffala. New York 14263

ABSTRACT

Liposomes containing polyethylene glycol-derivatized phospholipids

are able to evade the reticuloendothelial system and thereby remain incirculation for prolonged periods. We report here that doxorubicin encapsulated in these Sterically stabilized liposomes (S-DOX) suppresses the

growth of established human lung tumor xenografts in severe combinedimmunodeficient (SCID) mice and inhibits the spontaneous métastasesofthese tumors. The enhanced therapeutic efficacy of S-DOX compared to

free doxorubicin was demonstrated in two independent human/mousemodels. In the first model, S-DOX inhibited the growth of a humannon-small cell lung tumor xenograft established orthotopically in the lungsof SCID mice. Treatment of these mice with S-DOX, but not with free

drug, suppressed the growth of the tumor in the lung, prevented metastasis from the lung, and enhanced survival percentage. In another model,the human lung tumor is engrafted into the gonadal fat pad of SCID mice.Human tumor xenografts grow floridly in this site of engraftment, and thetumor spreads from this primary site into the peritoneal cavity andsubsequently reaches the liver and lung. In this model, free drug suppressed the growth of the primary tumor but had no effect upon thesubsequent spread of the tumor into the peritoneal cavity, liver, and lung.In contrast, treatment of the tumor-bearing mice with S-DOX (but not

with doxorubicin in conventional liposomes) suppressed the tumor spreadto the peritoneal cavity, completely arrested metastasis to the liver andlung, and suppressed the growth of the primary tumor xenograft. Thisreport provides the first evidence that antitumor drugs delivered bySterically stabilized liposomes can arrest the metastasis of human tumorxenografts.

INTRODUCTION

Sterically stabilized liposomes containing components such asphosphatidyl inositol, monosialoganglioside, or PEG-DSPE4 remain

in circulation for prolonged periods of time compared to other negatively charged liposomes (1, 2). These novel liposomes have beenused to deliver chemotherapeutic agents such as doxorubicin, epiru-

bicin, and vincristine to mouse colon or mammary tumors and shownto have enhanced therapeutic activity compared to the same drugsadministered in the free form or entrapped in conventional (non-longcirculating) liposomes (3-6). Previous studies have also shown en

hanced therapeutic effects of Sterically stabilized liposome drug delivery to human lung tumor xenografts in SCID mice (7) and humanprostate (8) and ovarian (9) tumor xenografts in nude mice. All ofthese previous studies have been limited to monitoring drug-liposome

Received 12/1/95; accepted 6/12/96.The costs of publication of this article were defrayed ¡npart by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by Grants CA54491 and CA57974 awarded by the

National Cancer Institute. United States Department of Health. Education and Welfare.2 Current affiliation: SEQUUS Pharmaceuticals. Inc., Menlo Park. California 94025.1To whom requests for reprints should be addressed, at Department of Molecular

Immunology, Roswell Park Cancer Institute. Elm and Carlton Streets. Buffalo. NY 14263.4 The abbreviations used are: PEG-DSPE, polyethylene glycol derivatized distearoyl

phosphatidylethanolamine; S-DOX. Sterically stabilized liposomes containing doxorubicin; C-DOX, conventional liposomes containing doxorubicin: F-DOX. free doxorubicin:SCID, severe combined immunodeficient: GFP. gonadal fat pad.

effects upon human tumors growing in nonorthotopic sites (i.e., s.c.),and there has been no evaluation in these previous studies of theability of the liposome drug delivery to prevent tumor metastasis. Wereport here for the first time the therapeutic effects of S-DOX upon the

growth of primary human lung tumors orthotopically engrafted withinthe lungs of SCID mice. We also report here the ability of thesedrug-containing liposomes to prevent the spread of human tumor

xenografts in two different human/SCID mouse models.

MATERIALS AND METHODS

Mice. CB17 seid/seid mice were obtained from our breeding colony atRoswell Park Cancer Institute. Our initial breeding stock was provided by Dr.Anne Croy (University of Guelph, Guelph, Ontario, Canada) with permissionfrom Dr. Mel Bosma (Fox Chase Cancer Center, Philadelphia. PA). SCID micewere housed in sterile microisolator cages (Lab Products, Maywood, NJ)without any prophylactic antibiotic treatments. All food, water, and beddingwere autoclaved before use, and all manipulations were done under sterileconditions in a laminar flow hood. Periodic screening of our user colony for thepresence of mouse pathogens is performed by challenging specific pathogen-

free C3H sentinel mice with soiled bedding and tissue from random SCIDmice. Four weeks after challenge, serum from sentinel mice is screened forantibodies to 16 different mouse pathogens (Mouse Level II Complete Antibody Profile, Microbiological Associates. Rockville, MD). CBI7+/+ controlmice were purchased from Taconic Laboratories (Germantown, NY) andhoused with conventional mice.

Doxorubicin Preparation and Injection. A long-circulating liposome formulation of doxorubicin (S-DOX) was prepared and characterized at Liposome

Technology, Inc., Menlo Park, CA. Although details of the methodology forpreparation of Doxil have been published (4), an outline will be given here. Anethanol solution of hydrogenated soy phosphatidylcholine (Natterman Phospholipids, Cologne, Germany), cholesterol (Croda, Fullerton, CA). PEG-DSPE, and ¿//-a-tocopherol (Hoffman LaRoche, Nutley, NJ) in a molar ratio of56.1:38.2:5.5:0.2 was injected into unbuffered 155 mM ammonium sulfate-lm,Mdesierai to a final concentration of 10% ethanol and shaken at 60°Cfor 1 h.

The liposome suspension was extruded through a sequence of nucleoporefilters from 0.4 f¿mdown to 0.05 /am diameter; it was then dialyzed against10% sucrose and the pH was adjusted to 5.5. Fifty fiM/ml of the liposomesuspension were mixed with 6.66 mg/ml doxorubicin (Farmitalia, Milan. Italy)in 10% sucrose at 60°Cfor 1 h. The sulfate salt, which is water insoluble,

forms as the doxorubicin enters the liposomes during preparation, and thedoxorubicin within the liposomes is in a semi-solid (gel) form (10). The

loading process and theory behind it have been reported previously (11).Nonencapsulated doxorubicin was removed, after cooling, by stirring theliposome suspension with Dowex 50W cation exchange resin. Final doxorubicin and lipid concentrations were 1.8 mg/ml and 19.5 /iM/ml, respectively.

A conventional, non-long circulating liposome formulation of doxorubicin-C-DOX, composed of phosphatidylglycerol, phosphatidylcholine, cholesterol,and dl-a-tocopherol in a molar ratio of 1:4:3:0.02 was prepared using thethin-lipid hydration method as described previously (12). Final doxorubicin

and lipid concentrations were 0.8 mg/ml and 35 ¿iM/ml.respectively.F-DOX, in powder form (Farmitalia), was solubilized in sterile saline to 2.0

mg/ml.The above preparations of doxorubicin were diluted in sterile saline for

injection into the lateral tail veins of SCID mice at the indicated doses.Injections contained total volumes of 50-150 ^1.

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Fig. 1. Orthotopic engraflment of human lung tumors in SCID mice. A. lungs of SCIDmice 7 weeks after inoculation with 1 X 10ft2E9 tumor cells transfected with a selectable

plasmici vector containing the histochemical marker gene of E. coli /3-galactosidase(LacZ). The tissue was stained for LacZ as previously reported (14). B. histológica!section of lung stained for LacZ revealing presence of the human tumors (a/rmr.c) in thelung of a SCID mouse inoculated with 1 X IO6 2E9 tumor cells 3 weeks previously.

Doxorubicin, both as free drug and in liposomes, was used at a dosage of 1.5mg/kg. This dosage was determined previously to represent the highest tolerated dosage of free drug in CB17 SCID mice (7). Conventional CB17 micetolerate at least 6 times this dosage. The increased sensitivity of CB17 SCIDmice is thought to be due to its intrinsic deficit in cellular mechanismsresponsible tor DNA repair.

Human Lung Tumor Cell Culture and Engraftment into SCID Mice. Afreshly resected squamous cell carcinoma of the lung (Department of ThoracicSurgery. Roswell Park Cancer Institute) was adapted to growth in vitro asfollows. Tumor tissue was debrided of necrotic tissue and minced with scissorsinto 3-mm' fragments. The fragments were then transferred to a triple-enzyme

preparation consisting of 2 mg/ml collagenase type IV (Worthington Biochemical Corp.. Freehold. NJ). 0.3 mg/ml DNase type I (Sigma Chemical Corp.. St.Louis. MO), and 5 /¿I/mlhyaluronidase (Sigma) in RPMI 1640 (Life Technologies, Inc.. Grand Island. NY). The tumor was digested overnight at roomtemperature with gentle stirring. The tumor cell suspension was collected,washed with RPMI 16406104. and plated onto sterile tissue culture flasks(Falcon 3024) in Dulbecco's modified Eagle's/F12 medium (Life Technolo

gies, Inc.) containing 57r fetal bovine serum, penicillin, and streptomycin. Amonolayer of Matrigel (Collaborative Research. Bedford. MA) 1 mm thickwas added to the plates on the third day of culture and allowed to stand for 4weeks. Medium was aspirated and replaced twice weekly. After initiation ofcell growth, the Matrigel was digested using dispase (Collaborative Research),and fresh medium was added. The cell line, named TL-I. was propagated in

tissue culture by trypsinization of confluent culture plates and has beenpassaged more than 100 times.

The TL-1 parental tumor cell line was cloned by a limiting dilution protocol

in which microculture wells are seeded with 1 cell/well. Clones of tumor cells

were expanded and phenotyped for their expression of cell adhesion moleculesand for their growth and metastatic potential in SCID mice. As reportedpreviously, one of the clones from the parental TL-I line was shown to grow

rapidly and to metastasize in SCID mice (13). This clone, termed 2E9. wasused exclusively in the work reported here.

Orthotopic Tumor Engraftment. Orthotopic engraftment of the tumorwas established by injecting 1.0 x IO6 2E9 cells i.v. into SCID mice. Within

7 weeks after tumor inoculation, multiple tumor nodules in the lung areobserved grossly in all lobes of the lung (Fig. \A). and microscopically, thesenodules are squamous cell carcinomas that invade and displace normal lungtissue (Fig. IS). The tumors shown in Fig. 1 were transfected with a selectableplasmid vector containing the histochemical marker gene of E. coli ß-galac-

tosidase (lacZ) as reported previously ( 14). Metastasis of these tumors in thelung to the ovary, pancreas, bone marrow, and adrenal glands has beenobserved in up to 8Qc/cof the mice (13).

Tumor Engraftment into the GFP. The engraftment of 2E9 tumor cells inthe GFP of SCID mice was achieved by injecting 2.5 X IO6 viable 2E9 cells

directly into the exposed GFP. SCID mice were anesthetized by injecting 0.3ml of Avertin i.p. A 3-mm vertical incision was made in the right lower

abdominal area through to the peritoneum. The right portion of the GFP wasdrawn up through the incision, and tumor cells were injected using a 25 gauge'/2-inch needle (2.5 X IO6 cells in 0.1 ml of RPMI 1640). The GFP was then

replaced into the peritoneal cavity and the peritoneal incision was closed witha 6-0 nylon suture. The skin incision was closed by metal staples. After

surgery, the mice were warmed on a heating pad until they awoke. This surgerywas conducted aseplically, and no death of animals due to the surgicalprocedure was observed. Tumors grew rapidly in the GFP, reaching a diameterof up to 4 cm within 7 weeks, and rapidly spread to other sites outside of theGFP. All of the mice had multiple smaller tumor foci (typically 8-12) located

outside of the GFP within the peritoneal cavity, and up to 80% of the mice havegross evidence of tumor metastasis to the liver or lung by 7 weeks afterengraftment.

RESULTS

Effect of Doxorubicin and S-DOX upon the Growth of Human

Lung Tumors Engrafted Orthotopically into SCID Mice. Lungtumor cells (1 X IO6 2E9) were injected i.v. into SCID mice to

establish Orthotopic lung tumor xenografts. Therapy with eitherF-DOX or S-DOX (each at a dose of 1.5 mg/kg) was initiated 1 week

after tumor inoculation. This treatment was repeated weekly for 8weeks. We determined previously that this dose of free drug is themaximum tolerated by the CB17 SCID mutant mice (7). ConventionalCB17 mice tolerate at least a 6-fold higher dose of doxorubicin.

During this period, four out of six mice treated with free drug diedwith multiple foci of tumor in the lung (Table 1). In contrast, all of themice treated with doxorubicin-containing liposomes survived and

contained significantly fewer tumor nodules (1.0 ±0.4, compared to45.0 ±5 in the mice treated with free drug). No tumors were observedoutside of the lung in the drug-liposome-treated mice, whereas two of

Table 1 Effect (if F-DOX and S-DOX upon the growth of human tumors inoculatedintravenously into SCID mice

No. of tumornodules"GroupControl

F-DOX'S-DOX'In

lung17.4

±9.845.0 ±5.0

1.0 ±0.4Outside

lung4/6

2/60/6No.

of mice surviving''/

no. of miceinoculated5/6

2/66/6

" Mice were inoculated intravenously with I X 10 2E9 tumor cells 1 week before the

initiation of therapy. Number of tumor nodules observed grossly in the lung and outsidethe lung were determined 9 weeks after inoculation and are reported as the mean numberof nodules per mouse ±SE, where n = 6. P < 0.01 (Mann-Whitney Test, two-tailed)where group S-DOX was compared with either the control or the F-DOX group.

Surviving at 9 weeks after 2E9 tumor inoculation.Mice were given 1.5 mg/kg doxorubicin either as free drug or encapsulated within

sterically stabilized liposomes. Mice received the drug ! week after inoculation and onceper week for 8 weeks.

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Table 2 S-DOX is superior to F-DOX in the suppression of growth and metastasis of

human tumor .\enografts in SCID miceAll the tumor weights in this table are expressed as the mean ±SE (n = 5), and the

P value was calculated based on the Mann-Whitney test (two-tailed).

Weight of tumor(g)GroupConlrol

F-DOX''S-DOX''GFP

(primary)*5.98

±1.391.38 ±0.190.79 ±0.23''Peritoneal

cavity'1.92

±0.841.73 ±0.760.33 ±0.20*Metastatic

lesions"Liver1/52/5

0/5Lung2/51/50/5

" Number of metastatic lesions observed grossly in the liver and lung.* Weight of tumor mass within the GFP (i.e., the site of tumor inoculation).' Weight of tumor in peritoneal cavity outside of the GFP.'' Mice were treated with 1.5 mg/kg of either F-DOX or S-DOX once per week for 8

weeks. Therapy was initiated 1 week after tumor inoculation. Control mice received tumorbut not doxorubicin.

e P < 0.01 when this number is compared with that of control group; P = 0.0745 when

this number is compared with that of F-DOX group.fP < 0.05 when this number is compared with that of either control or F-DOX group.

six mice treated with free drug and four of six untreated control miceshowed gross evidence of tumor growth outside of the lung.

S-DOX Prevents the Spread and May Limit Metastasis of

Human Tumor Xenografts Implanted within the GFP of SCIDMice. The 2E9 tumor was implanted into the GFP, and 1 week later,mice were treated with either F-DOX or S-DOX at a dose of 1.5mg/kg. Free drug and drug-liposome complex were injected i.v. each

week for eight weeks. Control mice were injected with saline. Nineweeks after tumor inoculation, all mice were sacrificed and the primary tumors in the GFP and the secondary tumors growing as multiple nodules along the mesentery were removed and weighed. Theliver and lung of each animal was observed for gross evidence oftumor metastasis.

The data presented in Table 2 establish that although both free drug(F-DOX) and the drug-liposome complexes (S-DOX) suppressed thegrowth of the primary tumor (i.e., within the GFP), only the S-DOX

suppressed the spread of the tumor to the mesentery and to the liverand lung. The mean weight of the tumor nodules in the mesentery ofcontrol mice or mice treated with free drug was 5.2-5.8 times themean weight of the tumor nodules in mice that received S-DOX. And

whereas three of five control mice and three of five mice treated withfree drug had gross evidence of metastasis of the tumor to liver orlung, no evidence of metastasis was observed in the mice treated withthe drug-liposome complexes. These results suggest that in addition to

suppressing the primary tumor growth, sterically stabilized liposomeshelp to retard tumor spreading and may prevent organ métastases.Previous studies have suggested that drug-containing sterically stabi

lized liposomes were superior to conventional liposomes in suppressing the growth of primary tumors in the lung (7). To determine if thesterically stabilized liposomes were better than conventional liposomes in suppressing tumor spread and metastasis, these two liposomes containing doxorubicin were compared directly for their ability

to inhibit tumor growth and spreading from the GFP to the mesentery,liver, and lung.

Drug-containing Sterically Stabilized Liposomes Have a Better

Therapeutic Effect Than Conventional Liposomes. 2E9 tumorcells (2.5 X IO6) were injected directly into the GFP. Two weeks after

tumor inoculation, mice were treated with C-DOX or S-DOX. Mice

received the drug at a dose of 1.5 mg/kg once per week for 8 weeksinjected i.v. Control mice were injected i.v. with saline.

Ten weeks after tumor inoculation, all of the control mice had verylarge primary tumors and a substantial tumor burden outside of theGFP. Three of the control mice had evidence of tumor spreading toeither the liver or the lung. Whereas the C-DOX suppressed the

growth of the primary tumor, less suppression of the secondary tumorgrowth in the mesentery was observed, and tumor spread to the liverwas found in two of the five mice (Table 3). Drug-containing steri

cally stabilized liposomes completely suppressed the organ metastasis, and the mean weights of the primary and secondary tumors weresuppressed 84% and 86%, respectively, compared to the control. Agraphic display of these results is presented in Fig. 2. Note that theprimary tumors from mice treated with S-DOX (Fig. 2a, Lanes 1-5}are smaller than those from mice treated with C-DOX (Fig. 2a, Lanes6-10) and smaller than the control untreated mice (Fig. la. Lanes11-15). Note also that the secondary tumors found in the mesenteryare fewer and smaller in mice treated with S-DOX (Fig. 2b, Lanes1-5) compared to the C-DOX treated (Fig. 2b, Lanes 6-10) oruntreated control mice (Fig. 2b, Lanes 11-15). We conclude that thedrug-containing sterically stabilized liposomes, compared to conven

tional liposomes, have a superior therapeutic effect for the treatmentof human lung tumor xenografts and that the doxorubicin-loaded

» »1 •¿�

..v »--.»•••«aefett o

o

<ta - > »,, v »

1 7 3 4 5 6 7 8 9 IO 11 12 13 14 15

Fig. 2. Human lung tumor xenografts removed from SCID mice. a. primary tumorsremoved from the GFP; b, the tumors found in the peritoneal cavity from individual micetreated with 1.5 mg/kg of S-DOX (1-5), 1.5 mg/kg F-DOX (6-10). or control untreated(11-15) mice. The mean weights ±SD of the primary tumors in the GFP and from the

tumors in the peritoneal cavity are presented in Table 3. Therapy was initiated 2 weeksafter tumor inoculation.

Table 3 Comparison of C-DOX and S-DOX for doxorubicin 's ability lo suppress the growth and metastasis of human tumor xenografis in SCID mice

All tumor weights in this table are expressed as the mean ±SE (n = 5). P value between each two groups was calculated based on Mann-Whitney test (two-tailed).

Weight of tumor (g) Metastatic lesions"

GroupControlC-DOX''

S-DOX''GFP*9.05

±0.853.02±0.57

1.46 ±0.25P<0.001=0.054Peritonea]

cavity'4.73

±0.682.32±0.29

0.66 ±0.27P=0.024=0.006Liver1/52/5 0/5Lung2/51/50/5" Number of metastatic lesions observed grossly in the liver and lung.' Weight of tumor mass within the GFP (i.e.. the site of inoculation).' Weight of tumor in peritoneal cavity outside of the GFP.JMice were treated with 1.5 mg/kg of doxorubicin in either C-DOX or S-DOX once per week for 8 weeks. Therapy was initiated 2 weeks after tumor inoculation. Control mice

received tumor but not doxorubicin.

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TREATMENT OF TUMORS WITH DRUG-LOADED LIPOSOMES

sterically stabilized liposomes arrest tumor metastasis and suppressthe growth of the primary tumor and its secondary spread to theperitoneal cavity.

DISCUSSION

Using two different tumor xenograft protocols [i.e., SCID mouseorthotopic (lung) and GFP], it has been established that S-DOX

suppresses the growth of established tumor xenografts within the primarysite and prevents subsequent spread of these tumors to other tissue sitesand organs. Whereas free drug and drug in conventional liposomessuppressed the growth of the primary tumor (albeit to a much lesserdegree), these treatment protocols did not arrest tumor métastases.Theenhanced efficacy of S-DOX in preventing the spread of tumors is a

significant finding and a previously unrecognized characteristic of thesenovel vesicles. This finding was not unexpected because it has beenestablished previously that the incorporation of monosialogangliosidephosphatidylinositol or PEG-DSPE into these liposomes prolongs theircirculation times by at least 5-fold over conventional liposomes, therebyincreasing the half-time in the blood to 15-22 h in mice (3, 6) and up to

42 h in humans (15). This increase in circulation time coincides with adecrease in the uptake of the sterically stabilized liposomes by the organsof the reticuloendothelial system such as the liver, and spleen (6, 16). Itis postulated that the prolonged circulation times of the sterically stabilized liposomes is due to the highly hydrated groups that sterically hinderthe binding of plasma components to the surface of the vesicles. Thebinding of plasma proteins to conventional liposomes leads to theirdestabilization and disintegration or to opsonization and subsequentphagocytosis by macrophages of the reticuloendothelial system (17).

The enhanced efficacy of sterically stabilized liposomes may alsobe in part due to their enhanced ability to accumulate within solidtumors in vivo (2, 16). The microvascular permeability and interstitialpenetration of sterically stabilized liposomes in both normal s.c. tissueand a human adenocarcinoma xenograft were quantified by using thedorsal skin-fold chamber implanted in SCID mice and intravitalfluorescence microscopy (18). A significant increase in the extravas-

cular accumulation of liposomes was observed in tumors that could beobserved for up to 1 week after the administration of the liposomes tothe mice (18).

Several different laboratories have attempted to preferentially directliposomes to tumors by attaching antibodies to the surface of the drug-containing vesicles (19-23). However, the success of the immunospecific

delivery protocol to drugs in vivo using liposomes with conventionalformulations has been limited (24-26). Recently, doxorubicin-loaded

sterically stabilized immunospecific liposomes (specific for the HER2proto-oncogene) have been shown to be markedly and specifically cyto-toxic against HER2-overexpressing human breast cancer cells in vitro,and the anti-HER-2 sterically stabilized liposomes administered to SCID

mice bearing the human breast tumor xenograft were shown to home toand deliver doxorubicin to the tumor in vivo (27).

We conclude that doxorubicin-loaded sterically stabilized lipo

somes represent a promising protocol for suppressing human lungtumor métastasesand that the attachment of tumor-specific antibodies

may enhance the efficacy of this drug delivery vehicle. The previouslyrecognized overexpression of the ß,integrin very late antigen-2 onhuman non-small cell lung tumors (28) represents a potentially viable

target for a sterically stabilized immunospecific liposome in thetreatment of human lung cancer.

ACKNOWLEDGMENTS

We thank Cheryl Zuber for typing the manuscript and Robert Parsons for hisexcellent technical support of this work.

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1996;56:3743-3746. Cancer Res   Takashi Sakakibara, Fang-An Chen, Hisashi Kida, et al.   Lung Tumor XenograftsLiposomes at Suppressing Growth and Metastases of HumanSuperior to Free Drug or Drug-containing Conventional Doxorubicin Encapsulated in Sterically Stabilized Liposomes Is

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