[CANCER RESEARCH 52, 6576-6582, December 1, 1992]

Ex Vivo Differentiation Therapy as a Method of Leukemic Cell Purging in Murine

Bone Marrow Expansion Cultures 1

M a r c u s O. M u e n c h , 2 Z i b a Guy, and M a l c o l m A. S. M o o r e

James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT

We investigated the use of differentiation therapy as a method of purging bone marrow (BM) of leukemic cells in ex vlvo murine BM expansion cultures (A-cultures). In clonal cultures and in suspension cultures a combination of the differentiation-inducing agents granulo- cyte colony-stimulating factor (G-CSF), interleukin (IL)-6, and all- trans-retinoic acid (ATRA) was found to be most effective in inducing the differentiation of the murine myelomonocytic leukemic cell line WEHI 3B D § LacZ clone 2.8 (clone 2.8). Furthermore, we investigated the activity of a mutant form of IL-6, mutein, and found it to have a greater specific activity in cell proliferation assays and in a clone 2.8 differentiation assay than the native form of IL-6. Coculture of clone 2.8 and BM in IL-1 and kit-ligand-stimulated A-cultures showed that the added stimuli, G-CSF, mutein, and ATRA, decreased the expansion of leukemic cells. Mice transplanted with G-CSF, mutein, and ATRA- purged BM had an increased survival time relative to nonpurged con- trols. The addition of G-CSF, mutein, and ATRA to A-cultures did not result in any impairment of hematopoietic stem cells when measured 5 wk after transplantation.

INTRODUCTION

Observations that the transformed phenotype of malignant cells can be reversed have suggested differentiation therapy as a possible means of cancer therapy. A number of chemicals, vi- tamin derivatives, and growth factors are in preclinical or clin- ical testing as effective differentiation-inducing agents (1). The cytokine G-CSF, 3 a growth factor involved in the normal growth and maturation of neutrophils, has been shown to stim- ulate the differentiation of a number of transformed cells. The ability to promote the differentiation of the murine my- elomonocytic leukemic cell line WEHI 3B D § was among the activities of G-CSF that led to its identification, purification, and the subsequent cloning of its gene (2, 3). The murine M1 and human HL-60 hematopoietic cell lines as well as several primary human leukemias have also been shown to differentiate in response to G-CSF stimulation (4, 5). Another cytokine shown to promote the terminal differentiation of transformed cells is IL-6. The cell lines WEHI 3B D +, M1, and the human histiocytic cell line U937 were shown to differentiate along the monocyte lineage when stimulated by IL-6 (6-8). The

Received 6/11/92; accepted 9/23/92. The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by USPHS Grants CA-32516, CA-20194, and CA-23766 from the National Cancer Institute; DK 42639 and HL-46546 from the NIH; and the Gar Reichman Fund of the Cancer Research Institute.

2 To whom requests for reprints should be addressed, at DNAX Research Insti- tute of Molecular and Cellular Biology, 901 California Ave., Palo Alto, CA 94304- 1104.

3 The abbreviations used are: G-CSF, granulocyte colony-stimulating factor; ATRA, aU-trans-retinoic acid; BM, bone marrow; BMT, bone marrow transplant; CFU-C, colony-forming unit-culture; CFU-L, colony-forming unit-leukemia; CML, chronic myeloid leukemia; A, delta; FBS, fetal bovine serum; GM-CSF, granulocyte-macrophage colony-stimulating factor; HPP-CFC, high proliferative potential-colony-forming cells; IL, interleukin; IL-6 mu, mutein; IMDM, Iscove's modified Dulbecco's medium; KL, kit-ligand; LPP-CFC, low proliferative poten- tial-colony-forming cells; NBM, normal murine BM; rh, recombinant human; rm, recombinant murine; clone 2.8, WEHI 3B D + lacZ clone 2.8; 5-FU, 5-fluorouracil.

vitamin A derivative retinoic acid has been shown to be an effective differentiation-inducing agent for the myeloid cell lines WEHI 3B D + and HL-60 as well as for most primary human myeloid leukemias both in vivo and in vitro (9-13). A comparison of 13-cis-retinoic acid and ATRA on differentiating promyelocytlc leukemic blast cells in vitro demonstrated ATRA tO be 10-fold more effective than the cis-isomer of retinoic acid (14). The ability of various differentiation-inducing agents such as G-CSF, IL-6, and ATRA to slow or prevent the growth of malignant cells suggests the use of such agents in the treatment of cancer. Among the possible applications of differentiation- inducing agents is their potential use for the ex vivo purging of cancer cells from normal BM cells.

We have recently demonstrated that the short-term culturing of murine BM cells in suspension cultures, A-cultures, stimu- lated by IL-1 + IL-3, IL-1 + KL, or IL-1 + IL-6 + KL can dramatically increase the number of hematopoietic progenitors in these cultures (15). The expansion of BM in IL-1 + IL-3- stimulated A-cultures resulted in an enriched progenitor popu- lation that, when transplanted into lethally irradiated mice, accelerated the recovery of peripheral blood cell numbers rela- tive to mice transplanted with fresh BM (16). Furthermore, the expansion of progenitor cells in A-cultures stimulated by IL-1 + KL was shown to be greater than in A-cultures stimulated by IL-1 + IL-3. The expansion of BM in vitro did not significantly decrease the long-term survival of the host mice or the prolif- erative potential of the transplanted hematopoietic stem cells. 4 By combining BM expansion with purging protocols that elim- inate tumor cells, a rich source of cancer-free progenitors could be obtained for purposes of BMT.

Protocols for BM purging with cytotoxic compounds have been shown to be effective in the removal of malignant cells while sparing early hematopoietic progenitors. However, there is also precedence for BM purging protocols that does not rely on the use of chemical purging methods. The observation that the growth of CML cells was selected against in long-term BM cultures prompted the use of such cultures as a method of selecting for normal hematopoietic progenitors (17). Trans- plantation of patients with BM cells extensively depleted of CML cells by growth in long-term BM cultures resulted in a variable duration of leukemia-free engraftment in some of these patients (18). These results lay a foundation for the purging of BM in ex vivo cultures using physiological means for selecting normal BM progenitor/stem cells while selecting against the growth of malignant cells.

In this study we investigated the use of differentiation therapy as a method of BM purging in a murine model system. Using the myelomonocytic cell line WEHI 3B D + LacZ clone 2.8 (clone 2.8), a subclone of WEHI 3B D + cells, we tested the efficacy of ATRA, G-CSF, IL-6, and a mutant form of IL-6 (IL-6 mu) as leukemic cell-purging agents in clonal cultures and A-cultures. A combination of ATRA, G-CSF, and IL-6 mu was found to be most effective in differentiating clone 2.8 cells.

4 M. O. Muench, M. T. Firpo, and M. A. S. Moore, manuscript submitted for publication.

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Cocultures of benign murine hematopoiet ic cells and clone 2.8 cells in IL-1 + KL-st imulated BM expansion cultures in the

presence of A T R A + G-CSF + IL-6 mu resulted in reduced numbers of leukemic cells relative to A-cultures st imulated only

by IL-I + KL. Mice receiving B M T of these cocultures dem- onstrated an increased survival t ime when transplanted with cells treated by ex vivo differentiation therapy.

M A T E R I A L S A N D M E T H O D S

Mice. Female BALB/c mice were obtained from a colony main- tained at our institute or were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were of at least 8 wk of age before use. The mice were housed under laminar-flow conditions along side sentinel mice which were tested free of specific pathogens. Mice were provided with acidified and/or autoclaved drinking water.

Growth Factors. Unless otherwise noted, all growth factors were used at optimal concentrations for the growth of BM progenitors in clonogenic assays. Purified rhIL-l~ (IL-I), specific activity = 1.32 x l07 units/mg (Syntex Laboratories, Inc., Palo Alto, CA), was used at 100 units/ml. Unless otherwise noted, purified rhIL-6 (Genetics Institute, Cambridge, MA) was used at 20 ng/ml. Purified rh mutein (IL-6mu), a mutant form of IL-6, was kindly provided by Imclone Sys- tems, Inc. (New York, NY). The IL-6 mu differs from the native protein by a deletion of 22 N-terminal amino acids and by a replacement of the cyteines at positions 45 and 51 with serine residues (19). IL-6 m" was used at 10 ng/ml unless otherwise stated. Purified rmKL, also known as mast cell growth factor or stem cell factor, was kindly provided by Dr. Steven Gillis (Immunex Corporation, Seattle, WA) and was used at 20 ng/ml. Purified rhG-CSF (Amgen Biologicals, Thousand Oaks, CA) was used at 10 ng/ml. Purified rmGM-CSF was used at 1000 units/ml (Immunex). ATRA (Sigma Chemical Co., St. Louis, MO) was dissolved in dimethyl sulfoxide as a stock solution at 1 • 10 -4 M and was used at 1 x 10 -6 M. Partially purified conditioned medium from the WEHI 3B D- cell line, containing murine IL-3, was used at concentrations equiv- alent to 20 ng/ml of purified rmIL-3 as determined in a proliferation assay utilizing the 32D cell line.

WEHI 3B D + lacZ clone 2.8. We have recently described the cloning of a subclone of WEHI 3B D + containing the lacZ gene in a retroviral vector. The subclone has been named WEHI 3B D + lacZ clone 2.8; however, the abbreviated name, clone 2.8, will be used throughout this paper. Clone 2.8 has been shown to be similar to the parental cell line in in vitro growth characteristics, responses to cytokine stimulation, and growth as a leukemia in vivo (20).

Clone 2.8 were maintained in IMDM supplemented with 10% FBS (Gemini Bioproducts, Inc., Calabasas, CA) and 0.05 mg/ml of gentam- icin (Gibco, Grand Island, NY). Cultures of clone 2.8 were passaged every 2 days by diluting the cells 20-fold in fresh medium.

CFU-C Assays. All clonal cultures and liquid cultures of murine BM cells or clone 2.8 were done in culture medium consisting of IMDM supplemented with 200/0 FBS and 0.05 m g / m l of gentarnicin. Colony ~says of NBM were pcrformcd as previously described (15). Colonies of primitive hematopoietic progenitors were grown from BM harvested from mice 1 day or 4 days after a 150-mg/kg body weight i.v. injection of 5-FU (Day 1 or Day 4 5-FU BM). Assays of CFU-C from 5-FU BM were done using a double-layer agarose culture as previously described (15). After 12 days of growth these cultures were scored for highly cellular colonies with diameters of at least 0.5 mm, HPP-CFC, or LPP-CFC containing at least 50 cells (21).

The clonal assays of clone 2.8, CFU-L, were done using a cell con- centration of 300 cells/35-mm culture dish. The clone 2.8 cells were grown in culture medium containing 0.36% agarose (SeaPlaque; FMC, Rockland, ME) for 7 days at 37~ in a fully humidified atmosphere containing 5% CO2 in air. CFU-L were scored as compact colonies or diffuse colonies in which at least 50% of the cells in a colony have migrated from the center of the colony. The diffuse phenotype has been correlated with the presence of differentiated WEHI 3B D + cells (22).

Measurement of IL-6 Activity in the B9 Cell Proliferation Assay. The activities of IL-6 and IL-6 m" were measured using the IL-6-depen- dent murine hybridoma B9 (23). The proliferation assay was done in IMDM supplemented with 2% FBS and 0.05 mg/ml of gentamicin. The IL-6 samples were serially diluted, in triplicate, in flat-bottomed 96-well tissue culture plates. The B9 cells were seeded at 5000 cells per well in a final volume of 0.1 ml. After 3 days of growth at 37~ in a fully humidified atmosphere of 5% CO2 in air, the cultures were pulsed with 0.5 ~tCi of [3H]thymidine for 6 h. Proliferation of the B9 cells was measured by liquid scintillation counting of DNA-incorporated [3H]thymidine.

The A-Assay. The expansion of clone 2.8 or Day 1 5-FU BM cells in liquid suspension cultures, A-cultures, was performed as previously described (15). Briefly, 5 x 103 clone 2.8 or 2.5 x 105 Day 1 5-FU BM cells were grown in quadruplicate 1.0-ml cultures at 370C in a fully humidified atmosphere of 5% CO2 in air. After 7 days of growth, with or without cytokine stimulation, the A-cultured cells were assayed for secondary CFU-C or CFU-L growth as described above. The change in progenitor cell numbers, the A-value, during the liquid culture phase of the A-assay is calculated as the number of secondary CFU-C or CFU-L divided by the number of primary CFU-C or CFU-L. The primary CFU-C or CFU-L number was determined from the input population of clone 2.8 or Day 1 5-FU BM cells at the onset of the liquid culture.

Cytokine Purging of Clone 2.8 Cells in BM Expansion Cultures. The expansion of 1 x 107 Day 1 5-FU BM cells in A-cultures was done in a volume of 40 ml of culture medium in 75-cm 2 tissue culture flasks (16). All BM expansion cultures were stimulated with IL-1 + KL. The cul- tures were grown for 7 days at 37~ in a fully humidified atmosphere of 5% CO2 in air. A-cultured cells, both the nonadherent and the adherent cells, recovered by trypsinization, were washed in Earle's balanced salt solution supplemented with 10 mM 1-3-D-4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid (pH 7.2) (Gibco), 0.05 mg/ml of gentamicin, and 1.0 mg/ml of bovine serum albumin (Sigma Chemical Co.) and resuspended for BMT.

Irradiation and BMT of Mice. Mice were irradiated with 8.5 Gy from a dual ~37Cs 7-ray source at a dose rate of approximately 0.9 Gy/min. Cells from BM expansion cultures were injected i.v. 2 to 3 h after the final irradiation. Each mouse received expanded BM cells from the input of 1 • 106 Day 1 5-FU BM cells, the number of cells equivalent to one tenth of a 40-ml A-culture. The cells were trans- planted by i.v. injection in a volume of 0.2 ml.

Statistics. The results are presented as the mean +_ SE. Significance was determined using the paired two-way Student t test. The results were considered significant if P _< 0.05.

R E S U L T S

Differentiation of Clone 2.8 Cells by ATRA, G-CSF, and IL-6 in Clonal Cultures. The cytokines G-CSF and IL-6, as well as the vi tamin A derivative ATRA, have been shown to promote

the terminal differentiation of WEHI 3B D § cells in vitro (4, 6, 7, 13). We investigated the ability of IL-1, KL, G-CSF, IL-6, ATRA, and a combination of all five factors to st imulate the differentiation of clone 2.8 cells in a C F U - L assay (Fig. 1). Nei ther IL-1 nor KL had any significant effect on the number of differentiated (diffuse colonies) CFU-L, but IL-1 increased the number of undifferentiated (compact colonies) C F U - L relative to unst imulated control cultures (P = 0.02). The number of diffuse colonies was increased in cultures st imulated by G-CSF, IL-6, ATRA, and the five-factor combinat ion (P -< 0.05, except for A T R A were P -- 0.15). The number of compact colonies was noticeably decreased in cultures st imulated by IL-6, ATRA, and the five-factor combinat ion (P < 0.05). The dramatic reduction in overall colony numbers observed in cultures st imulated by IL-6, ATRA, and the five-factor combinat ion reflects on the ability of these factors to induce the terminal differentiation of clone 2.8 cells, thus resulting in an increased proport ion of

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200

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Fig. 1. Comparison of the effects of different differentiation-inducing agents on the differentiation of clone 2.8 cells. Control A-cultures were not stimulated by any agent. The fewest compact and diffuse CFU-L were detected in cultures stimulated by a combination of all five agents. Columns, mean of 3 to 6 experi- ments; bars, SE.

four experiments, only 0 to 7 undifferentiated C F U - L were detected per 300 clone 2.8 cells cultured in the presence of 1 • 10 -6 M A T R A (Figs. 1 and 2).

Studies on the growth of human CFU-C have demonstra ted that A T R A can enhance the growth of commit ted myeloid pro- genitors (11, 24). The results presented in Fig. 3 demonstrate a similar enhancement of CFU-C growth from normal murine BM cells st imulated by G M - C S F in the presence of 3 x 10 -9 to 1 x 10 -6 M ATRA. The total number of CFU-C from N B M increased 1.7-fold when st imulated by G M - C S F and 3 x 10 -6 M A T R A as compared to G M - C S F st imulat ion alone (P < 0.05). Primitive hematopoiet ic progenitors, obtained from Day 1 5-FU BM, showed augmented growth at low concentrat ions of A T R A and decreased growth at higher concentrat ions of A T R A (Fig. 3). The cultures of Day 1 5-FU BM cells were st imulated with the combinat ion IL-1 + KL + IL-6 + G-CSF. The total number of CFU-C, H P P - C F C plus LPP-CFC, from Day 1 5-FU BM did not increase in response to A T R A stimu- lation contrary to the observed increase in N B M cultures. The 1.6-fold increase in H P P - C F C numbers at 3 • 10 - s M A T R A was at the expense of L P P - C F C numbers, suggestive that A T R A may increase colony cellularity at lower concentrat ions. At the highest concentrat ion tested, 1 x 10 -6 M, A T R A de- creased the numbers of H P P - C F C and L P P - C F C to 61 and

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ATRA Concentration (M) Fig. 2. A dose titration of ATRA in clonal cultures of clone 2.8 cells. Unstim-

ulated cultures (0 M ATRA) were grown in culture medium alone. At the highest dose of ATRA tested, 1 x 10 -6 M, only diffuse colonies of clone 2.8 cells were detected. Points, mean of CFU-L numbers from 3 culture dishes; bars, SE.

diffuse colonies and a reduction in total proliferation of clone 2.8 cells in these cultures. Cultures st imulated by all five factors averaged the fewest total number of diffuse and compact colo- nies; however, the number of colonies was not significantly less than in IL-6- or ATRA-treated cultures.

Effects of Varying Concentrat ions of ATRA on Clone 2.8 a n d

B M Progenitors. To further unders tand the effect of ATRA on the growth of mal ignant and normal hematopoiet ic cells, a dose t i t rat ion of A T R A in cultures of clone 2.8, NBM, and Day 1 5-FU BM was conducted. The results in Fig. 2 demonstrate that, at a concentrat ion as low as 3 x 10 -9 M, A T R A could stimulate a significant increase in diffuse C F U - L relative to cultures of unst imulated clone 2.8 cells. The total number of C F U - L decreased with increasing concentrat ions of ATRA. In

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5-FU BM. Cultures of NBM were stimulated with GM-CSF alone (0 M ATRA) or with GM-CSF and varying doses of ATRA. Cultures of Day 1 5-FU BM were stimulated with IL-1 + KL + IL-6 + G-CSF alone (0 M ATRA) or in combination with varying doses of ATRA. ATRA potentiated the growth of CFU-C from NBM over the dose range tested, but decreased the number of CFU-C detected in cultures of Day 1 5-FU BM at the highest concentrations tested. Points, mean of CFU-C numbers from 3 culture dishes; bars, SE.

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37% of control values, respectively. In all further studies, ATRA was used at 1 x 10 -6 M, since this concentration resulted in a very low number of undifferentiated CFU-L.

Comparison of IL-6 and IL-6 m" Activities on the Growth of Clone 2.8 and BM Progenitors. In the course of these studies we have had the opportunity to study the activity of a newly genetically engineered form of IL-6. This novel form of IL-6 has been named mutein which we have chosen to abbreviate as IL-6 m. The first 22 N-terminal amino acids of human IL-6 have been deleted, and the cysteine residues at positions 45 and 51 have been substituted with serine residues in IL-6 mu (19). These changes in IL-6 m" have resulted in a molecule with greater specific activity as demonstrated in the growth of the IL-6-dependent hybridoma B9. The activity of IL-6 m~ was nearly 200-fold greater than IL-6 in stimulating the prolifera- tion of B9 cells (data not shown).

The growth of clone 2.8 cells in response to different con- centrations of IL-6 or IL-6 m" is shown in Fig. 4. The number of diffuse CFU-L increased in cultures stimulated with increasing, 1 to 20 ng/ml, amounts of IL-6. However, in this experiment the total number of CFU-L was not noticeably affected by stim- ulation with up to 20 ng/ml of IL-6. In a titration of IL-6 mu the total number of CFU-L was decreased down to 14% of the number detected in unstimulated cultures. The increased activ-

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differentiation of clone 2.8 cells. In parallel clonogenic cultures IL-6 mu was much more effective in differentiating clone 2.8 cells than the native form of IL-6. This increased differentiation-stimulating activity was evident in the disappearance of compact CFU-L and a total decrease in CFU-L numbers in cultures stimulated with 5 to 20 ng/ml of IL-6 m in contrast to cultures stimulated with IL-6. Points, mean of CFU-L numbers from 3 culture dishes; bars, SE.

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Fig. 5. Comparison of dose titrations of rhIL-6 and rhIL-6 mu in stimulating the growth of HPP-CFC in synergy with IL-3. Points, mean of colony numbers from 3 culture dishes; bars, SE. d4, Day 4.

ity of IL-6 m" in stimulating the differentiation of clone 2.8 cells was evident in the complete loss of compact CFU-L at a con- centration as low as 5 ng/ml of IL-6 mu. These results demon- strate that IL-6 mu is much more active in causing the differen- tiation and subsequent reduction in proliferation of clone 2.8 cells than IL-6.

The activities of IL-6 and IL-6 m" on murine BM cells were tested in an assay of synergism between IL-3 and IL-6 in the stimulation of HPP-CFC from Day 4 5-FU BM (15). The in- creased activity of IL-6 m, relative to IL-6, was less dramatic in promoting the growth of HPP-CFC as compared to the results from the B9 proliferation assay and the CFU-L assay (Fig. 5). About a 2-fold increase in activity was observed in BM cultures stimulated with IL-6 ran. A concentration of 10 ng/ml of IL-6 m" was used in the following studies, since at this concentration a plateau number of HPP-CFC were stimulated, and the greatest differentiation of clone 2.8 cells was observed.

Inhibition of Clone 2.8 Cell Growth by G-CSF, IL-6, and ATRA in A-Cultures. The effects of IL-1, KL, G-CSF, IL-6, ATRA, or a combination of all five factors on the expansion of clone 2.8 cell number and CFU-L number in short-term sus- pension cultures, A-cultures, were examined (Fig. 6). The addi- tion of G-CSF, IL-6, ATRA, or the combination of factors greatly reduced the expansion of clone 2.8 cells in A-cultures. The smallest expansions of CFU-L were observed in cultures stimulated by the five-factor combinations. The combination including IL-6 had a A-value of 72, and the combination in- cluding IL-6 m" (combination A) had a A-value of 35, represent- ing a 9- and 19-fold reduction, respectively, in CFU-L expan- sion relative to control cultures. Morphological examination of cells recovered from A-cultures showed that clone 2.8 cells re- covered from control cultures had a typical blast cell morphol- ogy characteristic of this cell line. Cells recovered from combi- nation A-treated cultures showed increases in size, the presence of extensive vacuoles in the cytoplasm and, in many cases, segmentation of the nucleus; these morphological changes are suggestive of differentiation along the granulocyte or macro- phage lineages (data not shown). These results demonstrate that treatment with differentiation-inducing agents can greatly inhibit the growth of clonogenic clone 2.8 cells in A-cultures relative to unstimulated control cultures.

Limited Differentiation of Clone 2.8 Cells in BM Expansion Cultures Containing G-CSF, IL-6 mu, and ATRA. We have recently shown that cytokine-driven BM expansion cultures

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2250

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Fig. 6. Comparison of the effects of different differentiation-inducing agents on the differentiation of clone 2.8 cells in the A-assay. Control A-cultures were unstimulated. The combination-treated cultures were stimulated by all five agents shown in the figure. IL-6 mu was substituted for IL-6 in combination A-treated cultures (combination , ) . The smallest expansion, A-value, of total cell numbers (top) or CFU-L (bottom) in A-cultures was in the combination A-treated group. Columns, mean of 1 to 4 experiments; bars, SE.

provide a rich source of progenitor cells for purposes of BMT (16). In the experiments shown in Table 1, A-cultures of Day 1 5-FU BM were stimulated by IL-I + KL or IL-1 + KL + G-CSF + IL-6 m" § ATRA to expand their numbers of progen- itors during 7 days of in vitro suspension culture before trans- plantation into lethally irradiated mice. All mice transplanted with A-cultured Day 1 5-FU BM, without the presence of clone 2.8 cells, survived for greater than 35 days post-BMT (Table 1, Experiment A). The ability of G-CSF + IL-6 mu + ATRA to

purge A-cultures of Day 1 5-FU BM inoculated with either 1 x 105 or 1 • 10 4 clone 2.8 cells was tested in two experiments (Table 1, Experiments A and B, respectively). The BM expan- sion cultures stimulated by the five-factor combination showed reduced numbers of CFU-L after A-culturing relative to A-cul- tures stimulated by IL-1 + KL. These reduced CFU-L numbers correlated with significant increased survival time of host mice (P < 0.001). Although the expansion of clone 2.8 cells in co- culture with Day 1 5-FU BM cells was less than observed in A-cultures of clone 2.8 cells alone, the ability of G-CSF + IL-6 m" + ATRA to reduce, but not eliminate, the numbers of clonogenic tumor cells was consistent with the results from A-cultures of clone 2.8 cells alone (Fig. 6).

The Addition of G-CSF, IL-6 mu, and ATRA to IL-1 + KL- stimulated BM Expansion Cultures Does Not Decrease the Pro- liferative Potential of BM Stem Cells. Important in any ex vivo

treatment of BM for purposes of BMT is the effect of the ex vivo manipulations on the hematopoietic stem cells responsible for BM reconstitution. We have previously shown that, at 5 wk post-BMT, normal peripheral blood cell numbers, hematopoi- etic tissue cellularities, and a population of mature BM CFU-C had recovered to normal values, but at this time point total mouse HPP-CFC numbers were depressed relative to nontrans- planted mice (16). All mice receiving BMT with BM expanded in either IL-1 + KL or the five-factor combination survived for 5 wk after BMT (Table 1). However, the effect of 1 • 10 - 6 M

ATRA, as well as G-CSF and IL-6 mu, on early hematopoietic progenitors in IL-1 + KL-stimulated A-cultures was unknown. The observed inhibitory effects of ATRA on the growth of HPP-CFC (Fig. 3) suggested that the proliferative capacity of stem cells may be compromised by exposure to ATRA in A-cul- ture. To test this hypothesis, mice transplanted with A-cultured BM, as well as nontransplanted controls, were treated with 5-FU 5 wk post-BMT. The total hematopoietic tissue cellular- ities, BM and spleen, of control mice and mice transplanted with A-cultured BM cells were nearly identical 1 day after purg- ing with 5-FU (data not shown). However, in assays of early BM progenitors, HPP-CFC, BMT recipients had at least a 10-fold reduction in total HPP-CFC numbers 5 wk post-BMT relative to control mice (Fig. 7). The total number of neither LPP-CFC nor HPP-CFC was decreased in mice transplanted with BM cells expanded in A-cultures stimulated by the five- factor combination compared to mice transplanted with IL-1 + KL A-cultured cells. These results suggest that the addition of G-CSF + IL-6 mu + ATRA to IL-1 + KL-stimulated BM ex- pansion cultures does not diminish the proliferative capacity of the early hematopoietic progenitors.

Table 1 Survival of mice transplanted with cocultures of clone 2.8 and Day 1 5-FU BM

No. of clone 2.8 A-Culture cells inoculated No. of CFU-L Fraction

Experiment stimulus a into the A-culture post A-culture b survival c

No. of days of survival

post BMT d

850 rads No BMT None None 0/5

A IL-1 + KL None None 8/8 A Combination (A) None None 8/8 A IL-1 + KL 1 x l0 s 5.2 x 104 + 1.3 x 104 0/8 A Combination (A) 1 x l0 s 2.4 • 102 + 4.0 x 101 0/8

B IL-I + KL 1 x 10 4 6.9 x l0 s _+ 5.2 • 10 4 0/15 B Combination (A) 1 x 10 4 2.0 x 10 4 _+ 1.2 x 10 a 0/15

13.0 --- 1.3

>35 >35

12.9 + 0.5 15.6 + 0.8 e

10.2 -+ 0.2 13.0 -+ 0.4 e

a The combination (A) stimulus contained IL~ + KL + IL-6 m" + G-CSF + ATRA. l, The results are presented as the mean + SE of colony numbers from 3 culture dishes. c The survival of host mice was followed for 5 wk. d The results are presented as the mean + SE. e p < 0.001 relative to mice, within the same experiment, transplanted with IL-1 + KL A-cultured BM containing clone 2.8 cells.

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10 5

o 10 4

0 u,. 0

o. ,,.I 1~

o I--

o =E

0 i.i. 0 r -t- im

o I -

10 2 10 5

10 4

10 3

10 2

BONE MARROW PURGING BY DIFFERENTIATION THERAPY

affecting different aspects of cellular metabolism are more ef- fective than single-agent therapy. Furthermore, the heterogene- ity of any tumor cell population decreases the likelihood that

any single agent can be effective in killing all the malignant cells

11 BM HPP-CFC I [ ] Splenic HPP-CFC I

101

No BMT IL- I+KL Combination*

Fig. 7. Assay of total hematopoietic tissue LPP-CFC and HPP-CFC numbers in mice 5 wk after BMT. Mice were transplanted with IL-1 + KL or combination A (combination ,k)-stimulated A-culture expanded BM cells. The combination A-treated cultures were stimulated by the five agents listed in the legend to Fig. 6. Three age-matched nontransplanted mice were used as a control. BM and splenic tissue were pooled from 8 mice in the BMT groups. Although total colony num- bers were reduced in the BMT mice, relative to nontransplanted mice, the total numbers of CFU-C were similar in the two BMT groups. Columns, mean of colony numbers from 3 culture dishes; bars, SE.

DISCUSSION

The murine leukemic cell line WEHI 3B D + LacZ clone 2.8, like the parental cell line and the original leukemia, has a trans- formed phenotype. However, these cells can be induced by a number of agents to terminally differentiate along the granulo- cytic or monocytic lineages. Utilizing clone 2.8 cells in a murine BMT model we have tested ex vivo differentiation therapy as a method of purging benign BM cells of malignant clone 2.8 cells. When clone 2.8 cells were cocultured with Day 1 5-FU BM cells in IL-1 + KL-stimulated A-cultures in the presence of a com- bination of differentiation-inducing agents, the leukemic cell burden was reduced after 7 days of growth relative to cocultures stimulated only by IL-1 + KL (Table 1). The addition of the potential differentiation-inducing agents G-CSF, IL-6 mu, and ATRA reduced the expansion of clone 2.8 cells in A-cultures (Fig. 6; Table 1), but failed to completely eliminate growth factor-independent clonogenic cells (CFU-L) from these cul- tures. Transplantation of A-culture-expanded clone 2.8 and Day 1 5-FU BM cells into lethally irradiated mice demon- strated that the reduced CFU-L numbers achieved by the addi- tion of G-CSF + IL-6 mu + ATRA to A-cultures resulted in a slight, but significant, increase in survival time of host mice (Table 1). These results suggest that ex vivo differentiation ther- apy may be beneficial in BM-purging protocols.

Treatment of cancers with chemotherapeutic agents, in vivo or in vitro, is often best when a combination of chemical agents is used. The additive or synergistic effects of multiple drugs

in a population. A recent study from our institute has demon- strated the increased effectiveness of combining two chemo- therapeutic agents along with monoclonal antibody depletion for the in vitro purging of BM of acute myelogenous leukemic cells (25). Likewise, in this report we have demonstrated the increased effectiveness of combining differentiation-inducing agents in BM purging. Although single agents such as IL-6, IL-6 mu, or ATRA could greatly reduce the number of CFU-L and often completely eliminate nondifferentiated colonies in a clonogenic assay (Figs. 1, 2, and 4), a combination of agents, G-CSF + IL-6 mu + ATRA, proved to be most effective in stimulating the differentiation of clone 2.8 cells (Fig. 6). Since differentiation therapy alone could not completely halt tumor cell growth in our model system, these results indicate that BM purging with differentiation-inducing agents may be best if fur- ther combined with chemical and/or immunological purging methods.

Mutein, rhIL-6 mu, a novel synthetic form of IL-6, was com- pared to rhIL-6 in a number of assays in this study (Figs. 4 and 5). The amino acid modifications made to IL-6 mu have resulted in a molecule with greater specific activity than the native form of IL-6. Mutein was 200-fold more active in stimulating the proliferation of the hybridoma B9. The ability of IL-6 mu to stimulate the differentiation of clone 2.8 cells was also greatly increased relative to native IL-6. However, the activity of IL-6 mu on HPP-CFC growth from Day 4 5-FU BM was only slightly increased in comparison to native IL-6. These disparate results could possibly be explained by increased sensitivity of B9 and clone 2.8 cells to IL-6, relative to HPP-CFC, due to higher IL-6 receptor numbers on B9 and clone 2.8 cells. As suggested in this study, the greater activity of IL-6 mu may be of clinical importance.

Transplantation of BM into lethally irradiated mice leads to a loss of hematopoietic proliferative capacity in the hosts as compared to non-BMT mice. This reduction in proliferative capacity can be measured as a loss in early progenitor numbers. A study by Adam and Rosendaal (26) and a recent report from our laboratory, using A-cultured BM as a source of donor BM (16), have demonstrated a loss of total HPP-CFC numbers in BMT recipients. This loss of HPP-CFC numbers in BMT re- cipients was also observed in this study 5 wk post-BMT (Fig. 7). A comparison of total HPP-CFC per mouse in recipients of A-cultured BM expanded by IL-1 + KL or IL-1 + KL + IL-6 mu + G-CSF + ATRA demonstrated no significant difference be- tween the two groups. These results suggest that despite the inhibitory effect of 1 x 10 6 M ATRA observed in clonal cultures of Day 1 5-FU BM (Fig. 3), the addition of ATRA had no observed detrimental effect on hematopoietic stem cells respon- sible for long-term BM reconstitution.

The use of differentiation-inducing agents to promote the terminal differentiation of transformed cells in in vitro BM cultures may be applicable to BM-purging protocols for pur- poses of autologous BMT. In this study we combined the stim- ulus of IL-I + KL with the differentiation-inducing stimulus of IL-6 mu + G-CSF + ATRA to promote the expansion of benign hematopoietic progenitors. The culturing of human BM cells in cytokine-stimulated cultures following chemical and/or immu- nological purging protocols could help to increase the number

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BONE MARROW PURGING BY DIFFERENTIATION THERAPY

of transplantable progenitors. The addition of differentiation- inducing agents, which do not adversely affect the growth of benign hematopoietic cells, to BM expansion cultures could provide an added selection against cancer cells.

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1992;52:6576-6582. Cancer Res   Marcus O. Muench, Ziba Guy and Malcolm A. S. Moore  Purging in Murine Bone Marrow Expansion Cultures

Differentiation Therapy as a Method of Leukemic CellEx Vivo

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