Cancer dormancy: Isolation and characterization of dormant

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

Cancer dormancy: Isolation and characterization of dormant lymphoma cells EITAN 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, Department

of 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 term used to describe a prolonged quiescent state in which tumor cells are present, but tumor progression is not clinically apparent. Although clinical examples of tumor dormancy abound, little is known regarding the mechanisms underlying this state. Here we utilize an antibody-induced dormancy model of an aggressive murine B-cell lymphoma (BCL1) and show that the induction of the dormant state is accompanied by dramatic changes in tumor cell morphology and cell cycle status. These data indicate the feasibllty of altering the malignant phenotype of transformed cells by specific signals originating at the cell surface, and they suggest new opportunities for therapeutic intervention in cancer.

colony received weekly intravenous injections of 50 ,.g of polyclonal mouse anti-BCLI-Id obtained from the ascites fluid 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 to naive recipients. Cytofluorometry. Rat monoclonal antibodies (mAbs) specific for the following mouse antigens were used (see ref. 3): BCL1-Id (6A5) (F. Stevenson, General Hospital, Southampton, 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 Type Culture Collection (ATCC)]; CD45R/Ly5/B220 [RA3-6B2, phycoerythrin (PE)-conjugated]; and Thy-1.2 (53-2.1, PEconjugated) (PharMingen, San Diego). Normal rat IgG was prepared 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 sorting were performed on a FACScan [up to five-parameter analysis only; Becton Dickinson Immunocytometry Systems (BDIS)] and/or a dual-laser FACStar Plus (up to six-parameter analysis and sorting; BDIS), as previously described (4). The fluorochromes FITC, PE, and cychrome (CyC) were used with the single argon ion laser-equipped FACScan, whereas FITC and PE were used in combination with allophycocyanin (APC) and Texas Red (TR) on the argon ion and dye laser-equipped FACStar Plus. Typically, 106 cells were initially incubated with an unconjugated rat mAb, washed in phosphate-buffered saline (PBS), and then stained with either an FITC- or a TR-conjugated anti-rat immunoglobulin. After residual anti-rat immunoglobulin binding sites had been blocked with normal rat serum, the cells were incubated with a biotinylated mAb, washed, and then incubated with fluorochrome (PE and/or FITC)-mAb conjugates and a PE-, APC-, or CyC-streptavidin conjugate. Samples were fixed in 0.5% paraformaldehyde (in PBS) and protected from light until examined. For determinations including cell cycle analysis, antibody-stained cells (FITC, PE, and CyC) were simultaneously fixed in 0.5% paraformaldehyde, permeabilized (0.5% Tween 20), and stained with the DNA-binding dye Hoechst 33342 (40 pug/ml; Sigma). After incubation overnight at 4°C, these samples were analyzed in a specially prepared FACStar Plus equipped with both UV and 488-nm excitation and a pulse processing unit. All analyses employed appropriate light-scatter gates to exclude nonviable cells. In

Clinical examples of tumor dormancy are numerous, including cases of melanoma and breast carcinoma, in which periods of clinical latency may last for. a decade or more. Hence, the mechanisms that underlie induction, maintenance, and loss of dormancy are of great clinical importance. Despite this consideration and the fundamental insights that the study of dormancy might give to the biology of cancer and growth control of cells, the topic has received surprisingly little attention (1). Indeed, virtually nothing is known about the cellular and molecular events that constitute clinical dormancy, including whether dormancy represents a balance between cell growth and cell death or whether the tumor cells are in cell cycle arrest. The isolation of dormant cancer cells and determination oftheir cell cycle status would address this fundamental issue. Here we describe the analysis of tumor dormancy at the cellular level, using a murine lymphoma model. BCL1, a highly malignant B-lineage tumor that arose spontaneously in an elderly BALB/c mouse (2), offers a number of advantages for the study of dormancy (3): (i) the surface immunoglobulin idiotype (Id) is a clonal marker and, hence, represents a tumor-specific antigen; (ii) the BCL1 surface immunoglobulin contains a rare A-chain type, A3, which is operationally equivalent to a tumor-specific antigen; (iii) the tumor can be adoptively transferred to syngeneic recipients with a single BCL1 cell; and (iv) the tumor grows primarily in the spleen, and splenic enlargement (splenomegaly) can be detected by palpation. This simple noninvasive procedure facilitates repeated observations on individual animals.

MATERIALS AND METHODS Immunization of Mice. Male BALB/c mice 8-12 weeks old from our own colony were immunized with repeated injections of BCL1 IgM conjugated to keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (3). Severe combined immunodeficiency (SCID) mice 8-12 weeks old from our

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

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1829

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Proc. Natl. Acad. Sci. USA 90 (1993)

Immunology: Yefenof et al.

combinations of mAb against A light chain, Thy-i, Ia (class II major histocompatibility complex), and MAC-1 (CD11b) and the two scatter parameters, we have identified such a unique cluster 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 immunization with BCL1 IgM is depicted in Fig. 1. In the animal immunized with the BCL1 IgM without tumor challenge (Id-immune mouse), 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 the light chain loci. These few A+ cells are both small and large as judged by scatter parameters (Middle Left), reflecting resting and activated states, respectively. In mice challenged with BCL1 cells in the absence of prior immunization (BCL1 tumor) or with immunization with an isotype-matched IgM of a different idiotype from BCL1 IgM, the tumor grows rapidly, and the spleen contains large numbers of Thy-l-, A+ tumor cells (Top Center). While these cells are not expressing endogenous K, many of them are positive for both K and A (Top Right) resulting from the binding of host derived K-containing anti-BCL, antibody to the tumor cells. The BCL1 tumor cells are all large (Top Left), reflecting their activated malignant state. When mice are immunized with purified BCL1 IgM and then challenged with tumor cells, a clinical state of tumor dormancy is induced. Analysis of splenocytes from these

cell cycle analyses, cell doublets and larger cell aggregates were excluded from analysis by appropriate gating on the pulse processor signals of the Hoechst emission. Data were collected in list mode and analyzed with PAINT-A-GATEPLUS software (BDIS).

RESULTS AND DISCUSSION Isolation of Dormant Lymphoma Cells (DLC). In nonimmune mice injected with 106 BCL1 cells, tumors grow rapidly and 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 to injection of tumor cells (3), 70o of mice survive beyond 2 months (mean of 140 days). Hence, we chose lack of splenomegaly at 60 days as an operational definition of dormancy. Tumor cells are present in approximately 95% of such mice as demonstrated by transfer of tumors to syngeneic recipients or regrowth of tumors at a later stage. Id' B cells are not detected by cell surface staining with anti-Id antibodies, presumably because endogenous anti-Id is masking the cell surface Id on the tumor cells from subsequent analysis. Nevertheless, we postulated that DLC would likely have a unique "signature" of physical and antigenic characteristics (qualitative and quantitative) which would distinguish them from normal splenocytes and allow their isolation by highresolution multiparameter flow cytometry (4). Indeed, using

BCL1 Tumor in BALB/c Spleen j a: 800