Proc. Natl. Acad. Sci. USAVol. 90, pp. 1829-1833, March 1993Immunology

Cancer dormancy: Isolation and characterization of dormantlymphoma cellsEITAN YEFENOF*, Louis J. PICKERt, RICHARD H. SCHEUERMANNt, THOMAS F. TUCKER*,ELLEN S. VITETTA*§, AND JONATHAN W. UHRt¶*Lautenberg Center of Immunology, Hebrew University-Hadassah Medical Center, Jerusalem, Israel; tLaboratory of Molecular Pathology, Departmentof Pathology, *Department of Microbiology, and Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas, TX 75235

Contributed by Jonathan W. Uhr, December 7, 1992

ABSTRACT "Tumor dormancy" is an operational termused to describe a prolonged quiescent state in which tumorcells are present, but tumor progression is not clinicallyapparent. Although clinical examples of tumor dormancyabound, little is known regarding the mechanisms underlyingthis state. Here we utilize an antibody-induced dormancymodel of an aggressive murine B-cell lymphoma (BCL1) andshow that the induction of the dormant state is accompanied bydramatic changes in tumor cell morphology and cell cyclestatus. These data indicate the feasibllty of altering the ma-lignant phenotype of transformed cells by specific signalsoriginating at the cell surface, and they suggest new opportu-nities for therapeutic intervention in cancer.

Clinical examples of tumor dormancy are numerous, includ-ing cases of melanoma and breast carcinoma, in whichperiods of clinical latency may last for. a decade or more.Hence, the mechanisms that underlie induction, mainte-nance, and loss ofdormancy are of great clinical importance.Despite this consideration and the fundamental insights thatthe study ofdormancy might give to the biology ofcancer andgrowth control of cells, the topic has received surprisinglylittle attention (1). Indeed, virtually nothing is known aboutthe cellular and molecular events that constitute clinicaldormancy, including whether dormancy represents a balancebetween cell growth and cell death or whether the tumor cellsare in cell cycle arrest. The isolation of dormant cancer cellsand determination oftheir cell cycle status would address thisfundamental issue.Here we describe the analysis of tumor dormancy at the

cellular level, using a murine lymphoma model. BCL1, ahighly malignant B-lineage tumor that arose spontaneously inan elderly BALB/c mouse (2), offers a number of advantagesfor the study ofdormancy (3): (i) the surface immunoglobulinidiotype (Id) is a clonal marker and, hence, represents atumor-specific antigen; (ii) the BCL1 surface immunoglobulincontains a rare A-chain type, A3, which is operationallyequivalent to a tumor-specific antigen; (iii) the tumor can beadoptively transferred to syngeneic recipients with a singleBCL1 cell; and (iv) the tumor grows primarily in the spleen,and splenic enlargement (splenomegaly) can be detected bypalpation. This simple noninvasive procedure facilitates re-peated observations on individual animals.

MATERIALS AND METHODSImmunization of Mice. Male BALB/c mice 8-12 weeks old

from our own colony were immunized with repeated injec-tions of BCL1 IgM conjugated to keyhole limpet hemocyanin(KLH) in complete Freund's adjuvant (3). Severe combinedimmunodeficiency (SCID) mice 8-12 weeks old from our

colony received weekly intravenous injections of 50 ,.g ofpolyclonal mouse anti-BCLI-Id obtained from the ascitesfluid of BCL,-Id-immunized BALB/c mice.BCLI. The tumor was maintained by serial passage of

splenocytes. Growth was determined by splenic palpation(3), cytofluorometric analysis, and transfer of splenocytes tonaive recipients.

Cytofluorometry. Rat monoclonal antibodies (mAbs) spe-cific for the following mouse antigens were used (see ref. 3):BCL1-Id (6A5) (F. Stevenson, General Hospital, Southamp-ton, U.K.); mouse A (B.1.1); mouse K (Zymed Laboratories);Thy-1.2 (30-H12); Pgpl (CD44; IM7 biotin-conjugated);MAC-1 (CD11b; M170, biotin-conjugated) [American TypeCulture Collection (ATCC)]; CD45R/Ly5/B220 [RA3-6B2,phycoerythrin (PE)-conjugated]; and Thy-1.2 (53-2.1, PE-conjugated) (PharMingen, San Diego). Normal rat IgG wasprepared by chromatography on DEAE-Sephadex A-50.Mouse anti-I-Ed [13/18, fluorescein isothiocyanate (FITC)-conjugated] and anti-I-Ad (AMS-32.1, biotin-conjugated)(ATCC) were used.

Multiparameter flow cytometric analysis and cell sortingwere performed on a FACScan [up to five-parameter analysisonly; Becton Dickinson Immunocytometry Systems (BDIS)]and/or a dual-laser FACStar Plus (up to six-parameter anal-ysis and sorting; BDIS), as previously described (4). Thefluorochromes FITC, PE, and cychrome (CyC) were usedwith the single argon ion laser-equipped FACScan, whereasFITC and PE were used in combination with allophycocyanin(APC) and Texas Red (TR) on the argon ion and dyelaser-equipped FACStar Plus. Typically, 106 cells were ini-tially incubated with an unconjugated rat mAb, washed inphosphate-buffered saline (PBS), and then stained with eitheran FITC- or a TR-conjugated anti-rat immunoglobulin. Afterresidual anti-rat immunoglobulin binding sites had beenblocked with normal rat serum, the cells were incubated witha biotinylated mAb, washed, and then incubated with fluo-rochrome (PE and/or FITC)-mAb conjugates and a PE-,APC-, or CyC-streptavidin conjugate. Samples were fixed in0.5% paraformaldehyde (in PBS) and protected from lightuntil examined. For determinations including cell cycle anal-ysis, antibody-stained cells (FITC, PE, and CyC) were si-multaneously fixed in 0.5% paraformaldehyde, permeabi-lized (0.5% Tween 20), and stained with the DNA-bindingdye Hoechst 33342 (40 pug/ml; Sigma). After incubationovernight at 4°C, these samples were analyzed in a speciallyprepared FACStar Plus equipped with both UV and 488-nmexcitation and a pulse processing unit. All analyses employedappropriate light-scatter gates to exclude nonviable cells. In

Abbreviations: CyC, cychrome; DLC, dormant lymphoma cells;FACS, fluorescence-activated cell sorter; FITC, fluorescein isothio-cyanate; Id, idiotype; mAb, monoclonal antibody; PE, phycoeryth-rin; SCID, severe combined immunodeficiency.To whom reprint requests should be addressed at: Department ofMicrobiology, University of Texas Southwestern Medical Center,5323 Harry Hines Boulevard, Dallas, TX 75235.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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1830 Immunology: Yefenof et al.

cell cycle analyses, cell doublets and larger cell aggregateswere excluded from analysis by appropriate gating on thepulse processor signals of the Hoechst emission. Data werecollected in list mode and analyzed with PAINT-A-GATEPLUSsoftware (BDIS).

RESULTS AND DISCUSSIONIsolation of Dormant Lymphoma Cells (DLC). In nonim-

mune mice injected with 106 BCL1 cells, tumors grow rapidlyand splenomegaly is detectable by palpation by 23-42 days;most animals succumb to advanced tumors within 2 months.However, if the mice are immunized with BCL1 IgM prior toinjection of tumor cells (3), 70o of mice survive beyond 2months (mean of 140 days). Hence, we chose lack of spleno-megaly at 60 days as an operational definition of dormancy.Tumor cells are present in approximately 95% of such miceas demonstrated by transfer oftumors to syngeneic recipientsor regrowth of tumors at a later stage. Id' B cells are notdetected by cell surface staining with anti-Id antibodies,presumably because endogenous anti-Id is masking the cellsurface Id on the tumor cells from subsequent analysis.Nevertheless, we postulated that DLC would likely have aunique "signature" of physical and antigenic characteristics(qualitative and quantitative) which would distinguish themfrom normal splenocytes and allow their isolation by high-resolution multiparameter flow cytometry (4). Indeed, using

BCL1 Tumor in BALB/c Spleen

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combinations ofmAb against A light chain, Thy-i, Ia (class IImajor histocompatibility complex), and MAC-1 (CD11b) andthe two scatter parameters, we have identified such a uniquecluster of cells in spleens from mice with clinical dormancy.Flow cytometric analysis identifying DLC in the spleen of

an animal challenged with BCL1 cells following immunizationwith BCL1 IgM is depicted in Fig. 1. In the animal immunizedwith the BCL1 IgM without tumor challenge (Id-immunemouse), only 0.36% of splenocytes express the A light chain(Thy-l-, A+; Fig. 1 Middle Center). Essentially all of the A+cells are K (Middle Right), reflecting isotype exclusion at thelight chain loci. These few A+ cells are both small and largeas judged by scatter parameters (Middle Left), reflectingresting and activated states, respectively. In mice challengedwith BCL1 cells in the absence of prior immunization (BCL1tumor) or with immunization with an isotype-matched IgM ofa different idiotype from BCL1 IgM, the tumor grows rapidly,and the spleen contains large numbers of Thy-l-, A+ tumorcells (Top Center). While these cells are not expressingendogenous K, many of them are positive for both K and A(Top Right) resulting from the binding of host derived K-con-taining anti-BCL, antibody to the tumor cells. The BCL1tumor cells are all large (Top Left), reflecting their activatedmalignant state.When mice are immunized with purified BCL1 IgM and

then challenged with tumor cells, a clinical state of tumordormancy is induced. Analysis of splenocytes from these

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FIG. 1. Flow cytometric identification of DLC. Splenocytes from Id-immune control mice (Middle), Id-immune mice with DLC (Bottom),and nonimmunized mice bearing growing BCL1 tumors (Top) were analyzed for their light-scatter profile (Left), their expression of Thy-i vs.A (Center), and K VS. A (Right). For each plot 104 events are shown. In Left and Center the A+/Thy-l population is red and the remaining cellsare gray. In Right the A+ population is light blue or violet, delineating the K+ or K- subset, respectively.

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animals reveals a relatively large increase in the number ofThy-l1, A+ cells (Fig. 1 Bottom Middle; averaging 1.5 +

0.25% in 10 mice as compared with 0.24 ± 0.09% in 7 normalId-immune mice). These A+ cells are uniformly Ia+ MAC-1-(not shown), and more than 90% are K+ (Bottom Right). Thislatter finding suggests that they are coated with endogenousanti-Id and supports the interpretation that few of these cellsare normal A+ B cells. Indeed, multiparameter flow cytomet-ric analysis utilizing a panel of mAbs confirms that 290% ofthe cells within the A+ cluster are phenotypically distinct fromnormal B cells. Most of these putative DLC have the scatterprofile of small lymphocytes, reflecting an inactive state(Bottom Left). Thus, in clinically stable Id-immune micechallenged with malignant BCL1 cells 60 or more days priorto analysis, 1-2 x 106 quiescent tumor cells are present in thespleen, and they can be readily identified by using theparameters described above.To confirm that the (Thy-l-, Ia+) A+ cells delineated by

flow cytometric analysis are DLC, the tumorigenic potentialof these cells was examined in adoptive transfer experimentsafter their purification on the fluorescence-activated cellsorter (FACS). (Thy-l1, Ia+) A- and (Thy-l1, Ia+) A+ cellswere sorted from a mouse harboring DLC and compared withcontrol unseparated splenocytes from the same mouse andsorted (Thy-li, Ia+) A+ BCL1 tumor cells (from an unimmu-nized mouse with progressive splenomegaly) for their abilityto transfer tumors to syngeneic recipients. This was readilyaccomplished with sorted actively growing BCL1 cells (Table1). Unsorted splenocytes from mice harboring DLC alsotransferred tumors, but only in a portion of the recipientmice. Significantly, equivalent numbers ofthe sorted A+ cellstransferred tumors to all recipients. Titration results in indi-vidual experiments indicate significant enrichment of cellscapable of transferring tumors in the sorted A+ population ascompared with total splenocytes. Importantly, in none of thethree experiments performed (15 mice total) were we able totransfer tumors with the sorted A- population. These dataconfirm the identification of the DLC in the spleens of theseanimals.

Cell Cycle Status of DLC. The cell cycle status of BCL1 indormant and tumor-bearing mice was determined by usingmultiparameter flow cytometry and the DNA-binding dyeHoechst 33342 (5). Fig. 2A depicts the light scatter and DNAprofiles of splenocytes gated as A+, Ia+, and Thy-i-. Intumor-bearing mice, 17.6% ± 5.9%o ofthe growing BCL1 cellsare in the S (light blue) or G2 + M (black) compartments ofthe cell cycle. In contrast, only 1.8% ± 0.6% of the DLC arein S + G2 + M. The DLC are mostly smaller than thecorresponding population of Go + G1 cells from the BCL1tumor (violet), suggesting that the vast majority of the DLCare probably in Go. The BCL1 cells with a ln DNA contentare probably in G1, prepared to initiate the next cycle.Nevertheless, there appears to be a small portion of DLCwhich is cycling. Since the number ofDLC does not increasefor long periods of time, the low growth of the DLC must be

balanced by cell death. Whether such death is programmedfrom within or reflects the action of host mechanisms,immune or otherwise, remains to be determined.Morphology of DLC. The difference in characteristics

between growing BCL1 and DLC is also reflected in cellularmorphology. BCL1 tumor cells are large malignant-appearinglymphoid cells with abundant cytoplasm, large nuclei, mul-tiple prominent nucleoli, and open reticular chromatin (Fig.2B). In contrast, the majority of DLC are small and displaynuclei with clumped chromatin and absent or inconspicuousnucleoli (Fig. 2C). A minority ofDLC are medium-sized andexhibit larger, more actively appearing nuclei (Fig. 2C, solidarrow) and some DLC have a plasmacytoid appearance (Fig.2C, open arrow); however, these are still clearly distinguish-able from BCL1 cells of growing tumors.Mechanism of Dormancy Induction. The above data do not

indicate which branch(es) of the immune system, cellular orhumoral, is necessary for induction of dormancy. To addressthis issue, we have examined the induction of dormancy inSCID mice, which lack both T and B cells. We found that 18of 19 SCID mice developed dormancy after receiving weeklyinjections of polyclonal murine anti-Id antibodies (50 .g perinjection) over a period of 4-11 weeks and challenged with 3x 104 BCL1 intraperitoneally after the first injection ofantibody. FACS analysis of splenocytes from SCID mice,control or containing DLC, is depicted in Fig. 3. In normalSCID mice, no A+ cells are found. The small number ofThy-1+ cells probably reflects the presence of natural killer(NK) cells in the SCID spleen. A SCID animal with dormanttumor was sacrificed 70 days after challenge with BCL1 andanalyzed for the presence of tumor cells. A distinct popula-tion ofA+ cells was present in the spleen ofthis animal. Thesecells were the only cells which also stained with an anti-Kreagent (data not shown), indicating that the BCL1 cells hadbound the passively administered anti-Id antibody and thatno normal B cells arising from the SCID host were present inthe spleen. Hence, antibody without T cells is able to inducea clinical state of dormancy. This finding extends the obser-vations ofGeorge et al. (6) that injection ofanti-Id into normalmice later challenged with BCL1 could induce dormancy. Ourresult does not exclude the possibility that cellular immunityplays a role in the BALB/c Id-immune model discussedpreviously, but it indicates that humoral immunity may besufficient. Indeed, in a different murine lymphoma model ofdormancy, Wheelock (1) has shown that specific T lympho-cytes and macrophages that kill tumor cells in the peritonealcavity are the critical effector mechanisms. The use of theSCID model will allow the investigation of the interactionbetween cellular and humoral immunity in the induction oftumor dormancy in a well-defined system.

Loss of Dormancy in Id-Immune Mice. BALB/c miceprogressively lose dormancy as evidenced by the appearanceof splenomegaly and increasing numbers of Id+ spleen cellsindistinguishable by flow cytometry from growing BCL,

Table 1. Transfer of BCL1 disease from DLCStatus of tumor No. of cells transferred No. of mice with splenomegaly/

in donor Sorted for into recipients total no. of mice*

Control BCL, -t 104 13/13Control BCL1 A+ 104 14/14Dormant t 104 7/14

103 0/9Dormant A+ 104 14/14Dormant A+ 103 4/14Dormant A-, Thy-l- 104 0/15*Data compiled from three separate experiments; mice were last examined on days 171-313 aftertransfer.

tCells were exposed to antibodies and passed through the FACS without sorting.

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FIG. 2. (A) Cell cycle analysis ofDLC and BCL1 tumor cells. Splenocytes from Id-immune mice with DLC and nonimmunized mice bearingclinically progressing BCL1 tumors were simultaneously analyzed for their light-scatter proffle, DNA content (Hoechst 33342 staining), andexpression of A, Thy-1, and Ia. We collected 4300 events gated on (i) the A+/Thy-li/Ia+ population and (ii) the width vs. area of the Hoechst33342 signal (pulse analysis), so as to eliminate doublets and larger cell aggregates. The scatter and DNA profiles of the gated cells are shownin the left and right, respectively. Cells in the Go/G1 phase of the cell cycle (ln DNA content) are violet, those in S phase (between the 1- and2n peaks) are light blue, and those in G2/M phase (2n DNA) are black. (B and C) Morphology of growing BCL1 tumor cells (B) and DLC (C).Splenocytes from Id-immune mice with DLC, and nonimmunized mice bearing growing BCL1 tumor were stained for their expression of A,Thy-i, and Ia, and the A+/Thy-lV/Ia+ populations were sorted, cytocentrifuged onto slides, fixed with methanol, and stained withWright/Geimsa stain. (x500.) Arrows are discussed in the text.

cells. The rate of loss between 60 and 255 days after BCL,challenge is relatively constant (Fig. 4).These kinetics suggest that escape may be due to a single

stochastically determined event, perhaps mutation. DLCdescend from cells with a fully malignant phenotype andmajor abnormalities in karyotype; these genetically unstablecells have a high probability of further mutation. It would bepredicted that most of the mutations allowing the DLC toregrow (escapees) would not involve the Id. Thus, with

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polyclonal antibody holding DLC in check, several Idepitopes would need to be eliminated to render the antibodiesineffective. This prediction was verified by the finding that in21 of 27 mice that had lost dormancy, the escapees were Id+and transferred Id+ BCL1 tumors to recipients. The remain-

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ing 6 mice had low percentages of Id+, A+ cells in theirspleens, and these escapees could represent surface immu-noglobulin-negative variants.To determine whether escapees are mutants that are re-

sistant to induction of dormancy by Id immunity, escapeesfrom 3 mice (80 days after BCL1 challenge) were injected into31 Id-immune mice. Only 16% of the recipients becamedormant, as compared to 84% of Id-immune mice receivinggrowing BCL1 cells. These results indicate that the escapeeswere significantly less susceptible to the induction of dor-mancy than were wild-type BCL1 cells. Thus, escape fromdormancy may arise from a genetic change in the slowlyreplicating population in which the elements involving signaltransduction from surface immunoglobulin to the dormancyphenotype are mutationally altered. The idea that mutationalalterations in the DLC give rise to escapees is consistent withthe steady rate of dormancy loss that suggests a stochasticprocess. Nevertheless, our results do not exclude the possi-bility that changes in the host, such as waning of the immuneresponse, alteration in cellular effectors, or changes in cyto-kine levels, may also contribute to loss of dormancy.Hence, we provide evidence that the 3 BCL1 populations

under study-BCL1 tumor, DLC, and escapee-are eachdifferent. There are ligand-induced physiologic differencesbetween BCL1 tumor and DLC, whereas at least a portion ofthe escapees are genetically different. The most intriguingfinding is that a cell with a fully malignant phenotype can bebrought under growth restraint in vivo, and that restraint is soeffective that mutation of tumor cells may be a common, ifnot necessary, event for their escape.The question arises whether the BCL1 dormancy model

represents control of tumor growth by conventional immu-nologic effector mechanisms or whether it represents a moregeneralizable phenomenon of a tumor-specific agonist mim-icking a ligand-receptor interaction normally involved ingrowth or differentiation regulation. Evidence that specifictumor immunity plays a role in keeping cancer under controlcomes from a large array of experimental and clinical obser-vations, including the finding that immunosuppression cor-relates with both increased incidence and recurrence of sometypes of tumors (7). In the vast majority of experimentalmodels of anti-cancer immunotherapy, cellular immunity isthe major effector mechanism (8). Antibody-mediated immu-notherapy has been shown to be effective in a few instances-most notably the treatment of some cases of B lineagelymphoma (9)-but has not been efficacious in most epithe-lial tumors. It is not known whether any of the reported''successes" of immunotherapy are related to induction ofdormancy. Indeed, it is critical that a distinction be madebetween immune-mediated cytotoxicity (i.e., tumor cell kill-ing) and immune effector systems acting as agonist of growtharrest. Evidence for the agonist mechanism in our tumormodel is (i) BCL1 cells are present in substantial and stablenumbers in dormant spleen(s)-i.e., the tumor cells have notbeen destroyed by an active immune response; (ii) DLC arephysiologically different from growing BCL1 tumor cells; and(iii) escapees usually retain their tumor-specific antigen (Id)yet can be resistant to reinduction of dormancy.Taken together, these data suggest that the genetic alter-

ations leading to the changes in growth regulation and dif-ferentiation that characterize the malignant phenotype can bestably reversed by activation of the appropriate signal trans-duction pathways. It is likely that such signals act to "by-pass" or "override" the molecular "lesion" leading to themalignant phenotype and allow the cell to once again expressits normal phenotypic program. This state is precarious,however, since it involves a balance between the original

genetic lesion and the countermanding signals; additionalmutations or perhaps even alternative signals may shift thisbalance back towards the malignant phenotype.

In the BCL1 model, these countermanding signals appearto stem from binding of surface immunoglobulin by anti-Idantibodies, a natural signal transduction mechanism of B-lin-eage lymphocytes. In nonlymphoid cell types, other cellsurface molecules serve as links between the external envi-ronment and gene expression. Indeed, essentially all classesof cytokines and of cell or matrix adhesion molecules trans-duce signals from the outside of the cell to the nucleus andthereby influence all facets of cellular physiology. Thus, itwill be important to determine if the phenomenon of tumordormancy demonstrated here for a lymphoid malignancy isgeneralizable to other forms ofmalignancy-i.e., will tumorsof other histologic cell types express one or more cell surfacesignal transduction receptor(s) that have the potential tooverride their growth and differentiation defect? One exam-ple of such signalling may be the retinoic acid-induceddifferentiation of F9 teratoma cells, which is also associatedwith growth arrest (10). Indeed, the most extreme form of"dormancy" may be that observed when placement of te-ratocarcinoma cells into a normal blastula results in theincorporation of the tumor cells into the normal tissues of thedeveloped organism (11).The BCL1 model provides a useful paradigm for the

evaluation of tumor dormancy in humans. Using the analyt-ical techniques described in this report, it will be possible toevaluate naturally occurring human malignancies for thepresence of dormant cells and to study dormancy of humantumors in the SCID mouse model. Indeed, the demonstrationand characterization of dormant tumor cells in naturallyoccurring human tumors will be a critical test of the gener-alizability of the dormancy hypothesis.

Experimental findings from these models have implica-tions for the treatment ofcancer in humans. On one hand, theoccurrence of dormancy in human malignancies may impedetheir chemotherapeutic destruction, since most anti-canceragents preferentially act on cycling cells, and dormant cellsare predominantly resting. In such cases, the use of phar-maceuticals, such as immunotoxins, that can kill resting cells(12) may be required to cure the malignancy. On the otherhand, malignancies which are not amenable to standardtherapeutic protocols might be approached from the stand-point of dormancy induction rather than tumor destruction.

The valuable assistance ofDr. L. Terstappen, Ms. M. Nguyen, andMs. P. Collins in flow cytometry and the excellent secretarialassistance of Ms. C. Patterson are gratefully acknowledged. Thiswork was supported by a grant from the Tobacco Research Council.

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Cancer Res. 51, 5045s-5053s.4. Picker, L. J., Treer, J. R., Ferguson-Darnell, B., Collins, P. A.,

Buck, D. & Terstappen, L. W. M. M. (1993) J. Immunol., in press.5. Kubbies, M. & Friedl, R. (1985) Histochemistry 83, 133-137.6. George, A. J. T., Tutt, A. L. & Stevenson, F. K. (1987) J. Immu-

nol. 138, 628-634.7. Penn, I. & Brunson, M. E. (1988) Transplant. Proc. 20, 885-892.8. Greenberg, P. D. (1991) Adv. Immunol. 49, 281-355.9. Meeker, T. C., Lowder, J., Cleary, M. L., Stewart, S., Warnke, R.,

Sklar, J. & Levy, R. (1985) N. Engl. J. Med. 312, 1658-1665.10. Strickland, S. & Mahdavi, V. (1978) Cell 15, 393-403.11. Mintz, B. & IlMmensee, K. (1975) Proc. Natl. Acad. Sci. USA 72,

2585-2589.12. Vitetta, E. S., Krolick, K. A., Miyama-Inaba, M., Cushley, W. &

Uhr, J. W. (1983) Science 219, 644-650.

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