[CANCER RESEARCH 61, 333–338, January 1, 2001]

Apoptosis: An Early Event in Metastatic Inefficiency1

Christopher W. Wong, Andrea Lee, Lisa Shientag, Jong Yu, Yao Dong, Gary Kao, Abu B. Al-Mehdi,Eric J. Bernhard, and Ruth J. Muschel2

Departments of Pathology and Laboratory Medicine [C. W. W., A. L., L. S., J. Y., Y. D., R. J. M.], Radiation Oncology [G. K., E. J. B.], and Institute for Environmental Medicine[A. B. A-M.], University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT

Whereas large numbers of cells from a primary tumor may gain accessto the circulation, few of them will give rise to metastases. The mechanismof elimination of these tumor cells, often termed “metastatic inefficiency,”is poorly understood. In this study, we show that apoptosis in the lungswithin 1–2 days of introduction of the cells is an important component ofmetastatic inefficiency. First, we show that death of transformed, meta-static rat embryo cells occurred via apoptosis in the lungs 24–48 h afterinjection into the circulation. Second, we show that Bcl-2 overexpressionin these cells inhibited apoptosis in culture and also conferred resistanceto apoptosisin vivo in the lungs 24–48 h after injection. This inhibition ofapoptosis led to significantly more macroscopic metastases. Third, com-parison between the extent of apoptosis by a poorly metastatic cell line tothat by a highly metastatic cell line 24 h after injection in the lungsrevealed more apoptosis by the poorly metastatic cell line. These resultsindicate that apoptosis, which occurs at 24–48 h after hematogenousdissemination in the lungs is an important determinant of metastaticinefficiency. Although prior work has shown an association between ap-optosis in culture and metastasisin vivo, this work shows that apoptosisinvivo corresponds to decreased metastasisin vivo.

INTRODUCTION

Metastasis, the dissemination of tumor cells from a primary site todistant sites, is thought to occur by a series of steps in which tumorcells first migrate from the primary tumor, penetrate into the circula-tion, and eventually colonize distant sites. Evidence exists for each ofthese steps. Migration from the primary tumor site has been directlyobserved from tumors in the mammary fat pad, and intravasation fromtumors in the chicken chorioallantoic membrane has been demon-strated (1, 2). Entrance of cells into the blood stream can be observedvery rapidly after s.c. injections in experimental model systems andcan also be documented in human patients (3). Tumor cells can befound in the blood of cancer patients, although their presence oftendoes not predict prognosis (4–6).

Introducing tumor cells i.v. into immune-deficient experimentalmice can be used as a model to duplicate the later steps of hematog-enous metastasis for a wide variety of tumor cells. When lung coloniesdevelop after i.v. injection, the cell line is regarded as having meta-static potential. In general, lung colonies form only if the cells injectedare positive in spontaneous metastasis assays (i.e., if they form me-tastases after growth as s.c. tumors; Refs. 7 and 8). The injection ofmetastatic tumor cells into the mouse tail vein usually leads to theformation of lung, pleural, or mediastinal colonies, although coloniesat other sites sometimes occur. By harvesting colonies from specificsites and selecting through several cycles of injection, it has beenpossible to select for cells that preferentially metastasize to these sites,such as the ovary or liver (9). Homing to the lung can be partiallyattributed to the anatomy of the circulation that obliges all blood to

flow through the lungs. Some have proposed that cells lodge mechan-ically in the pulmonary capillaries due to size constraints, but capillaryocclusion cannot be sufficient in itself for growth as a metastasisbecause not all tumorigenic cells give rise to lung colonies (for reviewsee Refs. 10 and 11). Adhesion factors have been identified that arerequired for some tumor cells to attach to pulmonary endothelium. Forexample, adhesion of B16F10 melanoma cells in the lung has beenshown to depend on Lu-ECAM-1, whereas dipeptidyl peptidase IV(also known as CD26) is required for the attachment of R3230AC ratmammary and RPC-2 rat prostate carcinoma cells (12–16). Esb cellsderived from a murine T-cell lymphoma that metastasizes to the liverafter i.v. injection instead form colonies in the muscle whenb1integrin is disrupted, but only rarely form colonies in the lung (17).Furthermore, we have recently directly observed attachment of tumorcells to precapillary arterioles in the lung (18).

Whereas introduction of metastatic tumor cells into the circulationresults in colony formation, the majority of the injected tumor cells donot produce colonies. For example, i.v. injection of 53 104 cells mayresult in, at most, 200 colonies (19). The failure of the majority of thecells to form colonies has been termed “metastatic inefficiency” (20).Fidler and Nicolson (21) injected highly metastatic tumor cells labeledwith 125IUDR, a radioactive thymidine analogue, into mice. By meas-uring radioactivity levels in various organs, they were able to deter-mine where the injected cells migrated to after injection. Theseexperiments indicated that the majority of cells was rapidly clearedfrom the blood and initially arrested in the lungs (21, 22). Theydemonstrated that by 24 h,.85% of the cells initially arrested in thelungs were lost. Two cell lines were used, one with a 8–10-foldgreater ability to form pulmonary colonies than the other, yet both ledto equivalent counts retained in the lungs at 2 min to 1 day (9, 21).These observations have been confirmed with other cell lines and withdifferent methods of radioactive labeling by other groups (23–27).The fate of these cells that fail to metastasize has not previously beendemonstrated to be due to death by apoptosis.

To directly examine the fate of tumor cells within the lungs, welabeled metastatic tumor cells with GFP3 and observed their fate in thefirst 1–2 days after tail-vein injection. Our observations indicated thatmany of the injected cells undergo apoptosis within the lungs. Theseresults directly establish that tumor cells die in the lungs after intro-duction into the circulation through apoptotic processes.

MATERIALS AND METHODS

Cells and Cell Culture. The 2.10.10 and the 3.7 rat embryo fibroblast celllines are independentHras and v-myctransformed metastatic lines (28). RA3.1is a poorly metastatic rat embryo fibroblast cell line transformed byHras andE1A (29, 30). Clones overexpressing human Bcl-2 were isolated after trans-fection of 2.10.10 or 3.7 with pZIP-Bcl2 (kindly provided by John Reed,Burnham Institute, San Diego, CA) using lipofectamine (Life Technologies,Inc., Grand Island, NY), following the manufacturer’s directions. After selec-tion with 200mg/ml Geneticin (Life Technologies, Inc.), independent cloneswere isolated using cloning cylinders (2.10.10 Bcl-2 # 3; 2.10.10 Bcl-2 # 9).

Received 11/5/99; accepted 11/1/00.The costs of publication of this article were defrayed in part by the payment of page

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

1 Funded by National Cancer Institute Grant CA-46830.2 To whom requests for reprints should be addressed, at University of Pennsylvania,

269a John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104. Phone: (215)898-8401; Fax: (215) 573-4243; E-mail: muschel@mail.med.upenn.edu.

3 The abbreviations used are: GFP, green fluorescent protein; DAPI, 49,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediatednick end labeling.

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The cells were cultured at 37°C in 5% CO2 in DMEM (Life Technologies,Inc.) supplemented with penicillin-streptomycin (Life Technologies, Inc.) and5% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT). Selection wasmaintained throughout.

2.10.10 and HT1080 cells expressing GFP were isolated after transfection ofpEGFP-C2 (Clontech, Palo Alto, CA), 2 weeks of selection with Geneticin,followed by sorting in a flow cytometer. The 5% brightest cells were sortedand subcloned. This resulted in constitutively fluorescent clones. GFP-express-ing adenovirus was a kind gift from Meenhard Herlyn (Wistar Institute,Philadelphia, PA). B16F10 cells were infected with the virus at a ratio of 20plaque-forming units/cell, resulting in at least 90% fluorescent cells.

Radiation Treatment. Cells (13 106) were plated on 10-cm dishes. Thenext day, they were subjected to 10 Gy of Cesium 137 irradiation (ShephardMark I Model 68A irradiator). Twenty-four and 48 h following irradiation,cells were trypsinized, pelleted, and resuspended in 200ml of DMEM. Cells inthe medium and attached cells were pooled because apoptotic cells frequentlydetached. Ten microliters of propidium iodide solution [containing 3.4 mM

trisodium citrate, 0.1% NP40, 4.83 mM spermine tetrahydrochlorate, 0.5 mM

Tris, and 0.62 mM propidium iodide (pH 7.6)] was added to 20ml of cellsuspension, and the cells were examined under a fluorescent microscope. Theywere scored for apoptosis as determined by its morphology (ruffled edges,condensed chromatin and nuclear fragmentation, cell shrinkage, and formationof apoptotic bodies).

Tumorigenicity and Experimental Metastasis Assays.Female NCR-nu/numice, 4–6 weeks of age, were obtained from Taconic Farms (Germantown,NY) and housed aseptically (laboratory animal facilities, University of Pennsyl-vania). Cells used for injection were grown to subconfluence, subjected to brieftrypsin treatment, washed, and resuspended in serum-free DMEM. For tumori-genesis experiments, mice were injected bilaterally s.c. in both flanks with 53 105

cells in a single cell suspension in 100ml. Tumors were measured using verniercalipers for calculation of tumor size. Six tumors were measured for each timepoint. For experimental metastasis assays, mice were injected with a single cellsuspension of 13 104 cells in 100ml into the tail vein. Animals were killed whenexhibiting labored breathing or after 19 days. Lungs were fixed in 10% bufferedformalin, and a dissecting microscope was used to examine lungs for evidence ofmetastasis.

Intravital Video Microscopy. This assay was performed as described byAl-Mehdi et al. (18). Briefly, mice were sacrificed by an i.p. overdose ofsodium pentobarbitol at time points ranging from 30 min to 24 h after injectionwith cells expressing GFP. The chest was opened, and pulmonary circulationwas cleared of blood by gravity flow of perfusate through a cannula insertedin the main pulmonary artery, exiting from the transected left ventricle. Theperfusate was Krebs-Ringer bicarbonate solution [118.45 mM NaCl, 4.74 mM

KCl, 1.17 mM MgSO4.7H2O, 1.27 mM CaCl2.2H2O, 1.18 mM KH2PO4, 24.87mM NaHCO3 (pH 7.4), 10 mM glucose, and 5% dextran]. To visualize lungvasculature, the lungs were infused with DiI-acetylated low-density lipoprotein(Molecular Probes, Eugene, OR). The lungs were removed and examinedunder an inverted fluorescence microscope.

Fluorescent Microscopy. Ten-micrometer sections were cut from mouselung frozen in Histo Prep (Fisher Scientific, Fair Lawn, NJ). The sections werefixed in 2% paraformaldehyde and stained with 2.5mg/ml DAPI (SigmaChemical Co., St. Louis, MO). Green fluorescent cells were confirmed byoverlay with a DAPI-stained nucleus.

Immunohistochemistry. Ten-micrometer sections were cut from mouselung frozen in Histo Prep (Fisher Scientific). The sections were fixed in 2%paraformaldehyde and postfixed in 2:1 ethanol:acetic acid solution. A TrisNaCl buffer [1 M Tris, 140 M NaCl, and 0.1% Tween 20 (pH7.6)] was usedas rinsing buffer, and the sections were blocked with 5% goat serum. Theprimary antibody, polyclonal rabbit anti-GFP antibody, was obtained from theUniversity of Alberta, Calgary, Canada (Ref. 31; 1:1500; overnight at 4°C).The alkaline phosphatase-antirabbit IgG complex was from PharMingen (SanDiego, CA; 1:100; 1 h at room temperature). Levamisole (10 mg/ml; SigmaChemical Co.) blocked any endogenous alkaline phosphatase. The chromagenused to visualize the reaction was Stable Fast Red/Naphthol Phosphate (Re-search Genetics, Huntsville, AL), and the sections were counterstained withaqueous hematoxylin.

TUNEL Staining. Femalenu/nu mice, 4–8 weeks of age, were giveninjections with a single cell suspension of 23 106 cells in 100ml into the tailvein. After 24 or 48 h, the mice were sacrificed and the lungs were frozen in

Histo Prep. Ten-micrometer frozen sections were made, and apoptosis wasidentified using the ApopTag kit (Intergen, Gaithersburg, MD) according tothe manufacturer’s protocol. Briefly, sections were quenched in 2% hydrogenperoxide. The optimal dilution and incubation with the TdT enzyme was 1:54for 1.5 h at 37°C. We used an antidigoxigenin antibody from BoehringerManheim (Indianapolis, IN; 1:1000; 1 h at room temperature) in place of theantibody in the kit. The reaction was visualized by diaminobenzidine tetrahy-drochloride (Vector Laboratories, Burlingame, CA) as the chromogen, fol-lowed by a methyl green counterstain. Spleen sections were used as a positivecontrol with each preparation. The number of positive cells was determined bylight microscopy at3400 magnification. The slides were scanned using adigital micrometer (Microcode II; Boeckeler Instruments, Tucson, AZ) toensure that all areas were counted only once. To determine the area of thehistological sections, the slides were digitally scanned and the area of eachsection was calculated using Openlab software (Improvision, Coventry, UnitedKingdom).

Double-labeled apoptosis and anti-GFP sections were first stained withanti-GFP and visualized with Fast Red as described above, rinsed in water, andthen stained with the ApopTag kit and visualized by diaminobenzidine tetra-hydrochloride, as described above. The double-labeled sections were counter-stained with aqueous hemotoxylin.

Immunoblotting. Western blotting was as described by Maniatiset al.(32). Briefly, 106 cells were plated in 10-cm tissue culture dishes. The nextday, they were lysed with 200ml of sample buffer [10% glycerol, 2% SDS, 100mM DTT, and 50 mM Tris (pH 6.8)]. Protein samples were denatured byboiling for 5 min and run on a 12% SDS-polyacrylamide gel. After transferonto nitrocellulose membrane (Life Technologies, Inc.), the membrane wasblocked overnight in 5% milk-PBS. Bcl-2 was detected using a mouse anti-human Bcl-2 monoclonal antibody (Calbiochem, San Diego, CA) at a dilutionof 1:200 and a 1:5000 dilution of secondary antibody, goat antimouse IgGhorseradish peroxidase (Boehringer Mannheim). Bands were visualized usingthe enhanced chemiluminescence kit (Amersham Pharmacia, Piscataway, NJ).

RESULTS

Observation of GFP-labeled Tumor Cells in the Lungs after i.v.Injection. To directly observe the fate of tumor cells in the lungs aftertail-vein injection, we introduced a vector encoding enhanced GFPinto two metastatic sarcoma tumor cell lines: 2.10.10, a rat embryofibroblast transformed byHras and v-myc, and HT 1080, a cell linederived from a human fibrosarcoma (28, 33). Clones 2.10.10-GFP andHT1080-GFP, which express high levels of GFP, were isolated (18).2.10.10-GFP cells were injected i.v. into nude mice. After 24 h theanimals were sacrificed. Green fluorescent cells could readily bevisualized in frozen sections of the lungs (Fig. 1A). Counterstainingwith DAPI revealed the intact nuclei of the tumor cells surrounded bythe nuclei of the cells of the lung (Fig. 1B). We noted that many of thegreen fluorescent cells appeared to have ragged edges, often withblebbing of the surface, and that the intensity of the fluorescenceappeared diminished (Fig. 1C). The nuclei of these cells resembledapoptotic bodies (Fig. 1D). HT1080-GFP cells showed a similarmorphology after i.v. injection (data not shown; Ref. 18).

TUNEL staining was performed on the lung sections to determinewhether the morphological changes were associated with tumor cellapoptosis. TUNEL-positive cells could readily be detected asbrownafter staining (Fig. 2A). To show that the apoptotic cells were tumorcells, we used immunohistochemistry with an antibody to GFP toidentify the tumor cells. The anti-GFP antibody was visualized usingalkaline phosphatase-coupled anti IgG as a secondary antibody. Thedetection reaction for the alkaline phosphatase gives rise to aredcolor. GFP-positive cells could readily be detected in the frozensections from the lungs that had been injected with tumor cells, but notin the uninjected lungs (Fig. 2B). Double-staining revealed that manyof the TUNEL-positive cells also contained GFP, confirming thatapoptotic cells within the lung were, indeed, tumor cells (Fig. 2C). Insimilar experiments, other metastatic cell lines, including HT1080 and

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the murine melanoma B16F10, also gave rise to apoptotic cells within24 h of i.v. injection into nude mice (data not shown).

Effect of Bcl-2 on Apoptosis and Metastasis.An expressionvector for Bcl-2 was introduced into 2.10.10 and another clone oftransformed metastatic rat embryo cells, 3.7. Several clones thatexpressed Bcl-2 at markedly higher levels than the parental cells wereisolated (Fig. 3). These cells proved to be resistant to apoptosisinduced by radiation (Table 1). We then asked whether the number ofTUNEL-positive cells in the lungs differed after the injection of theparental cells compared with injection with cells overexpressingBcl-2. We counted the number of apoptotic cells in sections from thelungs harvested 24 or 48 h after i.v. injection of each cell type andcomputed the area of the sections counted (Table 2). Nine- to 11-foldfewer apoptotic cells were evident in the lungs of mice that receivedthe cells overexpressing Bcl-2 at 24 or 48 h (P 5 0.005). Thus, Bcl-2protected cells from apoptosis in the lungs following i.v. injection.The number of macroscopic lung colonies formed, on average, was5-fold greater (P# 0.001) after injection of either of two differentclones of the 2.10.10 cells expressing Bcl-2 relative to the parent line(Table 3).

Since Pietenpolet al. (34) had found that Bcl-2-overexpressingtumor cells sometimes have diminished tumorigenicity, we askedwhether tumorigenicity was affected in these cells. 2.10.10 cellsexpressing Bcl-2 or 2.10.10 parental cells resulted in equivalent tumorgrowth after s.c. injection into the flanks of mice (Fig. 4). Thus,

alteration in tumorigenicity would not seem to account for theseresults.

Difference in Apoptosisin Vivo between Cell Lines of DifferentMetastatic Potential. Since the results reported above suggest thatthe extent of apoptosisin vivo affects the outcome in the lungcolonization assay, we determined the numbers of apoptotic cellsfound in the lungs after injection of two different lines of transformedrat embryo fibroblasts with different metastatic potential. Transfor-mation of rat embryo fibroblasts byHras with either v- or c-mycoradenovirus E1A as cooperating oncogenes results in tumorigenicity.Cells transformed by ras plus E1A, such as the cell line RA3.1, areeither negative or only weakly metastatic in the lung colonizationassay. In contrast, rat embryo fibroblasts transformed byHras andmyc, such as 2.10.10, are highly metastatic in this assay (29, 33, 35).We compared both cell lines before injection intonu/nu mice andfound that,1% were apoptotic at the time of injection. Lungs werethen harvested 24 and 48 h after injection. Although both cell linesgave rise to apoptotic cells, injection of the poorly metastatic cell lineresulted in significantly more apoptotic cells, especially at the 24-htime point (P5 0.003; Fig. 5).

DISCUSSION

Our data indicate that apoptosis of tumor cells occurs in the lungswithin 24 h of i.v. injection and inhibition of that apoptosis canenhance lung colonization. Tumor cells that undergo less apoptosis inthe lungs were more likely to successfully establish colonies. Thiswork suggests that apoptosis is an important component of metastaticinefficiency, at early times after initial attachment. Our recent workusing intravital microscopy to examine tumor cells in the pulmonary

Fig. 1. Visualization of GFP-labeled tumor cellsin the lungs. 2.10.10-GFP (23 106) cells wereinjected i.v. intonu/nu mice. The lung was har-vested 24 h later, frozen in Histo Prep, sectioned,and counterstained with DAPI.A and C, DAPIstaining. B and D, green fluorescence.A and Bshow the same section, andC andD show the samesection.Arrows, tumor cells.

Fig. 2. Apoptotic 2.10.10-GFP cells in the lungs after i.v. injection. 2.10.10-GFP(2 3 106) cells were injected i.v. intonu/nu mice. After 24 h, lungs were harvested,sectioned, and stained either for apoptosis with the ApopTag kit (A), or for GFP asdescribed in “Materials and Methods” (B), or for both (C).

Fig. 3. Bcl-2 expression in transfected 2.10.10 and 3.7 clones. Lysates (60ml) fromeach of the indicated cells were immunoblotted using antihuman Bcl-2.

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circulation, has shown that circulating tumor cells attach to the pul-monary endothelium within 4 hin vivo or minutes in organ culture(18). By that time, all detected tumor cells are attached and no longerblood-borne (9, 18, 22, 36). Thus, the regulation of apoptosis must beinfluencing metastasis of tumor cells in the early stages after attach-ment to the pulmonary endothelium. The attachment of tumor cells ornormal cells to substratum has been shown in some instances toprovide survival signals (37, 38). These signals may be provided bybinding of integrins to extracellular matrix components (39, 40).Further downstream, activation of focal adhesion kinase has beenshown to provide protection from apoptosis induced by lack of adhe-sion (41). Apparently, the majority of the injected tumor cells, evenfrom a metastatic cell line will not encounter the survival signalsneeded to remain viable and proliferate. Whether survival signals areprovided through attachment to the endothelium, to extracellularmatrix components on the surface of the endothelial cells, or inexposed basement membrane or through secreted molecules is yet tobe established.

Survival in vivo in the circulation may be affected by mechanicalfactors. Although it has been proposed that cells are destroyed directlythrough sheer forces in the vasculature, it is also possible that me-chanical stresses lead to apoptosis in susceptible cells (42, 43). Theeffect of the immune system is also unclear. In these experiments,young nude mice were used to reduce the influence of immunereactivity to tumor cells. Furthermore, there is no reason to expect thatthe immune system would respond within 24 h or that differentialeffects would be seen between the various isogenic cells used in theseexperiments.

The expression of genes known to affect apoptosis in culture, such

as p53, Bcl-2, fas, integrina6, or nitric oxide production affectsmetastasis in experimental assays. Takaokaet al. (44) observed thatBcl-2 overexpression in B16 melanoma cells strongly enhanced pul-monary metastasis, but they did not examine the effect of Bcl-2 onapoptosisin vivo. Del Bufalo et al. (45) hypothesized that Bcl-2overexpression enhances tumorgenicity and metastatic potential ofMCF7ADR cells by inducing metastasis-associated proteins. How-ever, we found no significant differences in tumorgenicity by cellsoverexpressing Bcl-2 (Fig. 4). There are inverse correlations betweenthe extent of induction of apoptosis in culture and metastases. Thequestion raised by these studies is whether the signals used in culturebear any relationship to the actual signals received in animals. Niki-forov et al. (46) injected a mixture of two mouse fibroblasts, oneexpressing Bcl-2 and the other not expressing Bcl-2, into the tail veinof nude mice. They observed a significant enrichment of Bcl-2-

Table 1 Bcl-2 expression protects cells from irradiation-induced apoptosis

2.10.10 cells and those expressing Bcl-2, as shown in Fig. 3, were irradiated with 10Gy and harvested 24 and 48 h later.

Cell line24 h %

apoptotic48 h %

apoptotic

24 hUnirradiated% apoptotic

48 hUnirradiated% apoptotic

2.10.10 12.44 66.80 0.40 0.202.10.10 Bcl-2 #3 0.50 5.91 0.20 0.572.10.10 Bcl-2 #7 0.50 10.19 0.20 0.402.10.10 Bcl-2 #9 0.48 4.25 0.99 0.80

Table 2 Extent of apoptosis in the lungs after i.v. injection of tumor cells

Cells (23 106), as indicated, were injected i.v. into the tail vein ofnu/numice. After24 or 48 h, the mice were sacrificed and lungs were harvested, sectioned, and stained forapoptotic cells. The number of apoptotic cells on each section was counted, and the areaof the section was determined. Student’st test comparing Bcl-2 transfectants with parentalcells yieldedP 5 0.005.

Cell lines

Number of apoptotic cells/mm2

24 h 48 h

2.10.10 5.53 1021 NDa

2.10.10 Bcl-2 #7 0.483 1021 ND3.7 9.43 1021 8.23 1021

3.7 Bcl-2 1.03 1021 1.83 1021

a ND, not determined.

Table 3 Bcl-2 expression enhances metastasis

2.10.10 and transfectants #3 and #9 expressing Bcl-2 (13 104 cells) were injected intothe tail veins ofnu/numice (five mice each in the experiment shown). The animals weresacrificed when the mice exhibited labored breathing or at day 15, and the number of lungmetastasis was counted. Student’st test comparing transfectant #3 with 2.10.10 andtransfectant #9 with 2.10.10 yieldedP 5 0.0002 andP 5 0.001, respectively. A duplicateexperiment showed similar results.

Cell line2.10.10 Bcl-2

#32.10.10 Bcl-2

#9 2.10.10

Number of lung metastasis 89.86 25.91 476 14.14 14.86 2.8

Fig. 4. Bcl-2 expression does not affect growth of 2.10.10 cells as s.c. tumors. Cells(5 3 105) from 2.10.10, and two clones of 2.10.10 cells expressing Bcl-2, 2.10.10 Bcl-2# 7 and 2.10.10 Bcl-2 # 3, were injected s.c., and tumor size was measured (see “Materialsand Methods”). Student’st tests on day 13 data yielded the followingPs: between parentaland clone 3,P 5 0.74; between parental and clone 7,P 5 0.051; between parental andboth Bcl-2 clones,P 5 0.429.

Fig. 5. Comparison of the numbers of apoptotic cells in the lungs after injection ofhighly metastatic 2.10.10 or poorly metastatic RA3.1. 2.10.10-GFP or RA3.1-GFP(2 3 106) cells were injected i.v. intonu/numice. After 24 or 48 h, lungs were harvested,sectioned, and stained for apoptosis with the ApopTag kit. The number of ApopTag-positive cells was counted, and the area of the counted section was determined. At 24 h,P 5 0.003; at 48 h,P 5 0.132.

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expressing cells recovered 2 h after incubation in the lungs. At thesame time, PCR analysis of total lung DNA revealed no change in theratio of the injected cells. These data suggest that the depletion ofnon-Bcl-2-expressing cells was due to loss of viability rather thanescape from lungs. However, Bcl-2 overexpression failed to alter thefrequency of experimental metastasis in this case (46). McConkeyetal. (47) also reported that a nonmetastatic prostate carcinoma cell linewas more susceptible to apoptosis in tissue culture, when comparedwith a metastatic prostate carcinoma cell line that expressed twice asmuch Bcl-2. Owen-Schaubet al. (48) similarly found that melanomacells resistant to fas-mediated apoptosis were more prone to metasta-size, and that fas knockout mice had higher numbers of metastasis.Xie et al. (49) have demonstrated that nitric oxide production resultsin apoptosis in culture and that there is an inverse correlation betweenapoptosis in culture and metastasis. In this study, we extend theseresults examining apoptosis 24–48 hin vivo after injection and showthat Bcl-2 overexpression inhibits this early apoptosis. We alsoshowed that the difference in metastatic potential between two pairedcell lines correlated with the difference in apoptosisin vivo at 24–48h after injection. Thus, the effect is in addition to the possible effectson cell proliferation, dormancy, and apoptosis that will determine therate of nodule growth.

In other studies of metastatic inefficiency, Luzziet al. (50) ob-served only 20% cell death of B16F1 after injection into the superiormesenteric vein. They attributed metastatic inefficiency to the failureof dormant solitary cells to initiate growth and failure of early micro-metastases to continue growth into macroscopic tumors (50). Theseresults are significantly different with the results obtained after injec-tion of radiolabeled B16F1 in that;70–80% of radioactivity locatedin the liver is lost after 24 h, suggesting extensive cell loss (21, 22).Cameronet al. (51) observed only 25% cell loss in the lung of mice3 days after injection of B16F10 cells into the inferior vena cava, but75% cell loss after 3 days. Although this correlates with the study byLuzzi et al. (50), their observation regarding cell loss conflicts withthe observations of Fidler and Nicolson (21), also using B16F10 cellsand the same mouse strain, that.90% of cells are lost within 1 dayof injection (9, 22, 36, 50).

Additional data linking apoptosis to metastasis include studiesexamining the effect of CD44. Overexpression of a soluble CD44fragment in a metastatic murine mammary carcinoma cell lineblocked metastasis. The cells secreting this CD44 fragment underwentconsiderably more apoptosis in the lungs than the parental cells at24–48 h after injection (52).

The ability of apoptosis to modulate long-term growth of a meta-static tumor colony may be separate from the apoptotic regulation thatdetermines early survival (24–48 h) after attachment to the pulmo-nary endothelia. These studies indicate that the extent of apoptosisearly after pulmonary attachment correlates with the outcome in termsof formation of lung colonies.

In several cases, genes that affect apoptosis and metastasis havebeen found to affect the number of TUNEL staining cells when thecells were grown as tumors. Kimchi’s (53–55) laboratory isolated agene,DAP, that bears a death domain that can interact with signalsthrough fas or tumor necrosis factora and whose expression affectsthe growth of lung carcinoma cells as tumors or as metastases.Similarly, metastatic breast carcinoma cells in whicha6b1 het-erodimerization is prevented grow poorly as tumors with increasedapoptosis and have decreased lung colonization (56). In these cases,whether the effect is on the ability of the cells to grow as a tumor orwhether survival in the circulation is also affected has not beenestablished. Our methods allow a distinction between survival in thecirculation and later growth as a tumor. They could be applied to

directly evaluate the effect of specific genes on survival in the circu-lation.

ACKNOWLEDGMENTS

We are grateful for funding from the National Cancer Institute.

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2001;61:333-338. Cancer Res   Christopher W. Wong, Andrea Lee, Lisa Shientag, et al.   Apoptosis: An Early Event in Metastatic Inefficiency

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