In vitro differentiation and establishment of cell lines derived from human myelomonocytic leukemia cells

Immunology Letters, 12 (1986) 225-230 Elsevier Imlet 734

IN VITRO DIFFERENTIATION AND ESTABLISHMENT OF CELL LINES DERIVED FROM HUMAN MYELOMONOCYTIC LEUKEMIA CELLS* Abraham J. TREVES ~, Vivian BARAK l, Miriam H A L P E R I N 1, Shoshana BIRAN 1, Rachel LEIZEROWITZ 2, and Aaron POLLIACK 2 Departments of ~Radiotherapy and Clinical Oncology and 2Hematology, Hadassah University Hospital, Jerusalem, Israel 91120 (Received 21 November 1985) (Modified version received 23 December 1985) (Accepted 24 December 1985)

I. Summary Primary cultures of cells derived from 13 patients with acute myelomonocytic leukemia (AMML) were studied with particular emphasis on in vitro proliferation, cell differentiation and the mode for establishment of cell lines. Using irradiated human macrophage monolayers to assist cell growth, we obtained four new cell lines of myelomonocytic origin. All the cell lines were characterized for cytochemical markers and response to phorbol esters (TPA), a differentiation inducing agent. In the absence of any inducing agent, spontaneous differentiation of blast cells into mature macrophages-like cells occurred in 8 out of the 13 primary cultures. Thus, maturation induction by agents such as TPA is not always required in order to obtain leukemic cell differentiation in vitro. The regulation of cell proliferation and differentiation by cellular interactions and by extrinsic soluble products is discussed in detail, in the light of these findings.

differentiation and continue to multiply. Despite their unrestricted growth in vivo, attempts to cultivate these cells and establish growing cell lines in vitro have met with only limited success. Nevertheless, several human myelomonocytic cell lines have been established [1-8], and these have been used widely to study cell differentiation [9-11], macrophage functions [12, 13] and monokine secretion [5, 6, 14, 15]. In general, the incidence of success in the establishment of cell lines is low and unpredictable and the reasons for this failure are usually unclear. In our experience, culture deterioration and the development of lymphoblastoid cells appear to be the major causes. In the past, we have observed that cultured cells from patients with A M M L have matured spontaneously into macrophages [16]. Recently, we have exploited this phenomenon to increase the rate of success in establishing AMML-derived cell lines. The method for establishment of these cell lines and the characteristics of some new AMML lines are the subject of this paper.

2. Introduction Leukemic cells of the myelomonocytic lineage are probably arrested at certain stages of their Key words: myelomonocytic cells - differentiation - cell lines * Supported by a research grant from the Isreal Cancer Research Fund, New York.

3. Materials and Methods 3.1. Origin and &olation of cells Cells were obtained from the peripheral blood of 13 patients with A M M L at the time of diagnosis, prior to chemotherapy. Peripheral blood was also obtained from healthy volunteers to es-

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tablish normal macrophage monolayers. Venous blood was mixed with 50 IU/ml sodium heparin (Leo Pharmaceutical Products, Bellerup, Denmark), diluted 1:1 with Ca+2Mg+2-free phosphate-buffered saline (PBS, pH 7.2) and separated on Ficoll-Paque solution (Pharmacia, Uppsala, Sweden) as described [17]. The separated peripheral blood mononuclear cells (PBMC) were washed once with PBS and resuspended in culture medium (RPMI-1640) supplemented with MEM vitamin solution (1:100); penicillinstreptomycin, 50 u/ml and 50 mg/ml, respectively; 2 mM L-glutamine and 1 mM sodium pyruvate (Biological Industries, Beth Haemek, Israel). 3.2. Cell cultures PBMC from patients were plated at high cell density ( 5 - 1 0 x 106/ml) in culture medium supplemented with 15% fetal calf serum (FCS, Gibco, Grand Island, NY) and 5% heat-inactivated pooled human AB Rh-positive serum (HS) in 50 ml tissue culture flasks (Nunc, Roskilde, Denmark). The cultures were fed with fresh medium and split every 4 - 7 days for the first 2 - 3 wk. When cell proliferation ceased, part of the non-adherent cells were transferred to flasks coated with irradiated macrophages (described below). For the following 4 - 8 wk, the cultures were transferred to flasks containing macrophage monolayers. Later the cells were transferred to flasks lacking macrophage monolayers. Following the establishment of continuous growth, the cell cultures were split at a ratio of 1:4 every 4 - 8 days in 50-ml culture flasks. The cell lines M24 and M26 were considered as established lines after 8 mth from their first cultivation. The M20 cell line has already been described previously [16]. The M28 cell line has also passed the 8 mth period but eventually it was contaminated with mycoplasma and discharged. Macrophage monolayers from healthy donors were established by plotting 5 × 106 PBMC per 1 ml culture medium containing 2°70 FCS for 1 h, 5 ml in each 50-ml culture flask. The non-adherent cells were then removed by repeated washing and the adherent cells were further cultured for 7 days. The macrophages were then irradiated with 3000 rads using a 3' source irradiation mathin. 226

The HL60 cell line, established from a patient with acute promyelocytic leukemia [4] was kindly donated by T. R. Breitman, the National Cancer Institute, Bethesda, MD. All cultures were maintained at 37 °C in a humidified incubator containing 5°7o CO 2. 3.3. Morphological examination and function tests Cytocentrifuge preparations were stained with Wright's Giemsa stain for light microscopy. Cytochemical and other cell markers were also tested on cytospin preparations, as previously described [16]. Phagocytic activity was assessed by the ingestion of latex particles (0.8 ~tM bead diameter, Sigma, St. Louis, MO) after 24 h incubation, as in an earlier study [18]. Cells which internalized 5 or more latex beads were scored as positive for phagocytic activity. Cell-free supernatants obtained from 2×106 cells/ml of serumfree medium cultured for 3 days, were tested for lymphokine activities as described in previous studies [16-18]. 3.4. Treatment with TPA In the experiments described in Tables 2 and 3, 10 6 ceils were plated in multiwell Linbro plates and treated with 10 ng/ml of 12-O-tetradecanoyl phorbol 13-acetate (TPA, Sigma) for 24 h. Following incubation for 4 h with or without TPA, the number of adherent cells was counted and their proportion to non-adherent cells calculated [19]. The cells were also collected and cytospin slides were prepared for light microscopic, cytochemical and phagocytic studies.

4. Results

In this study we attempted to cultivate and establish cell lines from the PBMC of AMML patients. The experiments described were all performed under similar culture conditions, which according to preliminary experiments, yielded the longest continuous proliferation of AMMLderived ceils. During the first 2 - 3 wk of culture, ceils proliferated, but in 8 of the 13 cases some o f the cells ceased to proliferate, became adher-

Table 1 Cell lines and differentiation of primary cultured AMML-derived cellsa Expt. no.

Spontaneous differentiation~

Establishment of cell line

Reason for failure

M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30 M31 M32

yes yes yes yes yes no no yes yes no no no yes

yes no no no yes no yes no yes no no no no

development of LBCc development of LBCc culture deterioration culture deterioration culture deterioration culture deterioration culture deterioration culture deterioration culture deterioration

a AMML-derived cells from 13 cases were cultured in vitro as described in Materials and Methods. b Bottom of the flasks covered with a monolayer of mature macrophages capable of phagocytosing latex beads. Few non-adherent cells continue to proliferate. c LBC-lymphoblastoid cell line, EBNA-positive cells grown in large clumps. ent a n d u n d e r w e n t m a t u r a t i o n to m a c r o p h a g e s (Table 1). In these cases, a u n i f o r m m o n o l a y e r o f large, a d h e r e n t m a c r o p h a g e s c a p a b l e o f p h a g o c y t o s i n g latex b e a d s covered the b o t t o m s o f the flasks. W h e r e v e r a m a c r o p h a g e m o n o l a y e r developed, m o r e m a r k e d p r o l i f e r a t i o n o f the n o n - a d h e r e n t cells c o n t i n u e d to take place a n d for l o n g e r p e r i o d s o f time. However, the presence o f m a t u r e m a c r o p h a g e s d i d n o t always g u a r a n t e e the d e v e l o p m e n t o f a p e r m a n e n t cell line. Accordingly, we a d d e d i r r a d i a t e d m a c r o p h a g e s f r o m n o r m a l i n d i v i d u a l s to the cultures, in o r d e r to p r o v i d e a l o n g e r p e r i o d o f c o n t a c t between the p r o l i f e r a t i n g cells a n d the m a c r o p h a g e s . This t e c h n i q u e seemed to increase the v i a b i l i t y o f the n o n - a d h e r e n t cells a n d e n a b l e d t h e m to survive a n d proliferate d u r i n g the early critical p e r i o d o f the l o n g - t e r m cultures. In a d d i t i o n , the presence o f h u m a n s e r u m a n d m a c r o p h a g e s a p p e a r e d to prevent the d e v e l o p m e n t o f l y m p h o b l a s t o i d cells (LBC) which w o u l d o t h e r w i s e proliferate a n d take over the cultures in m o s t cases. In all cases in which the cells h a d n o t been t r a n s f e r r e d to m a c r o p h a g e m o n o l a y e r s (8 cases) the cells c o m pletely ceased to proliferate a n d no cell line was o b t a i n e d . In three o f the p r i m a r y cultures at-

t e m p t e d , s o m e o f the cells were c u l t u r e d in the presence o f 30°70 (v/v) m e d i u m c o n d i t i o n e d by m a t u r e m a c r o p h a g e s (7-day-old c o n d i t i o n e d m e d i u m ) . N o n e o f these cultures survived, ind i c a t i n g t h a t intact m a c r o p h a g e s were required for s u p p o r t i n g cell p r o l i f e r a t i o n . Thus, by emp l o y i n g the above d e s c r i b e d m e t h o d o l o g y we obt a i n e d f o u r new A M M L - d e r i v e d cell lines f r o m the 13 cases a t t e m p t e d (Table 1). Two o f the newly established cell lines were carefully c h a r a c t e r i z e d a n d c o m p a r e d to the one cell line a l r e a d y described, the M-20 [16] a n d the well established HL-60 cell line [4, 20]. U n d e r the light microscope, stained p r e p a r a t i o n s o f b o t h M24 a n d M26 cells revealed a mixed p o p u l a t i o n o f myeloblasts a n d promyelocytes which c o n t a i n e d c y t o p l a s m i c granules. T h e M26 seemed m o r e mature, consisting o f smaller cells with a lower n u c l e a r - c y t o p l a s m i c ratio. Very few cells f r o m all four cell lines tested were a d h e r e n t o r expressed p h a g o c y t i c activity. F o l l o w i n g treatm e n t with T P A for 24 h, all cell lines m a n i f e s t e d typical m a c r o p h a g e functions. S o m e o f the cells in each p o p u l a t i o n even a c q u i r e d the m o r p h o l o g ical a p p e a r a n c e o f m a c r o p h a g e s , b e c a m e adherent to plastic surfaces a n d p h a g o c y t o s e d latex 227

Table 2 Induction of cell differentiation by TPA a

Adherence (%) Morphology b Phagocytic activity (°70)

M20

M24

M26

HL60

42 +

31 _+

89 +

90 +

40

50

47

31

Percentages of positive cells following treatment with TPA. In the absence of TPA only a few cells from each cell line were either adherent or phagocytic. b The adherent cells were morphologically identified as either mature macrophages ( + ) or various stages between monocytes and macrophages (+_).

beads (Table 2). By these criteria, the M26 cells responsed better than the M24 cells to TPA treatment. Different results were obtained when the cells were tested cytochemically before and after treatment with TPA. Prior to incubation with TPA, both M24 and M26 cells were positive for chloroacetate esterase, myeloperoxidase, acid phosphatase and Sudan black, but only the M24 cell line was positive for nonspecific esterase. In contrast to the M20 cells, which responded to TPA by an increase in the number of cells containing nonspecific esterase and a decrease in myeloperoxidase [16], M24 and M26 cells showed little cytochemical change after incubation with TPA (Table 3). The only significant change was a decrease in the percentage of M24 cells positive for myeloperoxidase. Neither M24 nor M26 cells produced interleukin 1, interleukin 2 or colony stimulating factor Table 3 Cytochemical findings of cells before and after treatment with TPA ~ M24

Non-specific esterase A50-chloroacetate esterase Myeloperoxidase Acid phosphatase Sudan black

M26

-

+ TPA

87 58 61 97 97

99 58 49 97 94

+ TPA 8 95 85 92 95

8 97 93 97 98

°70 positive cells after counting 500 cells in each specimen

228

for granulocytes and/or macrophages, while the M20 cells secreted interleukin 1 [16]. All three cell lines were nonreactive for H L A - D R antigens, E B N A or surface Ig [16].

5. Discussion In the present report we describe a new method which increases the frequency of success of establishment of new cell lines of myelomonocytic origin. The method is based on the use of irradiated macrophage monolayers which assist the proliferating cells to overcome the critical period in culture and become independent in their continuous growth thereafter. It appears that the macrophage monolayers serve as efficient feeder layers for the growing cells, but other activities may also explain their role in this system. In this respect, it is indeed possible that macrophages may secrete factors which inhibit maturation and thus enhance continuous proliferation of immature cells and precursors. Macrophages also have been found to secrete iron binding proteins, interferons or prostaglandins [21-23]. Such factors were found to inhibit myelomonocytic colony formation in vitro [24-26]. In addition, we have recently reported that human macrophages secrete a factor which was inhibitory for in vitro monocyte maturation [27]. It is also possible that following their establishment the cell lines secrete factors which sustain their own continuous growth. In preliminary studies we found that M20 and G D M secrete a CSF-like factor that also induces enhanced proliferation in the other AMML-derived cell lines when cultured at suboptimal conditions. However, the exact mechanism by which macrophages enable the leukemic cells to proliferate in vitro is yet to be established. In preliminary experiments the presence of human serum in the culture medium seemed to inhibit the development of LBC. This phenomenon may be due to the presence of antibodies against the EBV, which always is associated with the induction of LBC [28]. In 4 of the 13 cases, successful establishment of permanent cell lines was achieved. In an earlier study, the M20 line was

shown to be o f m y e l o m o n o c y t i c origin [16] a n d the newly established M24 a n d M26 cell lines possess similar characteristics. M o s t o f the cells in b o t h cell lines were f o u n d to c o n t a i n chlor o a c e t a t e esterase, m y e l o p e r o x i d a s e a n d acid p h o s p h a t a s e a n d to stain positively for S u d a n b l a c k (Table 3). T h e M24 cell line c o n t a i n e d n o n specific esterase a n d is p r o b a b l y m y e l o m o n o c y t i c in n a t u r e while the M26 lacks this m o n o c y t i c esterase a n d a p p e a r s to be m o r e myeloblastic. B o t h cell lines were negative for E B N A a n d surface Ig. T h e response to T P A in each o f the three cell lines was different (Tables 2 a n d 3 a n d [16]). T h e o n l y c o m m o n features a p p e a r e d to be the expression o f plastic a d h e r e n c e a n d p h a g o c y t i c activity following t r e a t m e n t with T P A (Table 2). T h e M20 cells r e s p o n d e d to T P A with a decrease in the a m o u n t s o f c h l o r o a c e t a t e esterase a n d mye l o p e r o x i d a s e a n d an increase in n o n - s p e c i f i c esterase activities [16], i n d i c a t i n g t h a t these cells were i n d u c e d to m o n o c y t i c d i f f e r e n t i a t i o n while this was less evident in the M24 a n d M26 cell lines. O t h e r cell lines o f similar origin, such as H L 6 0 , U937, T H P I a n d G D M , also r e s p o n d e d to T P A with increased a d h e r e n c e a n d p h a g o c y t i c activity [16]. Thus, each cell line m a y represent a different stage o f cell d i f f e r e n t i a t i o n a n d show a different p a t t e r n o f responsiveness to differentiat i o n - i n d u c i n g agents. T h e q u e s t i o n o f d i f f e r e n t i a t i o n i n d u c t i o n in m y e l o m o n o c y t i c cell lines has been carefully investigated [9-11]. However, o u r finding o f ' s p o n t a n e o u s ' d i f f e r e n t i a t i o n in 8 o f the 13 prim a r y cultures raises a d d i t i o n a l questions. T h e u n u s u a l f i n d i n g t h a t m o s t o f the surviving cells in such p r i m a r y cultures differentiate into m a t u r e m a c r o p h a g e - l i k e cells, suggests t h a t this process is i n h i b i t e d in vivo, while the established cell lines o b t a i n e d m a y represent a selective p o p u l a t i o n o f cells which are u n a b l e to m a t u r e a n d thus c o n t i n u e to proliferate. W h a t role these p h e n o m e n a p l a y a n d w h a t effect soluble regulat o r y factors p l a y in leukemic cell growth in vivo remains to be established. In o t h e r systems, ' s p o n t a n e o u s ' m a t u r a t i o n o f p r o l i f e r a t i n g i m m a t u r e cells to m a c r o p h a g e - l i k e cells has been reported. C o n t i n u o u s p r o l i f e r a t i o n o f m o n o c y t i c cells in the presence o f v i t a m i n D

a n d h y d r o c o r t i s o n e was always a c c o m p a n i e d by m a t u r a t i o n to m a c r o p h a g e s by s o m e o f the cells [29, 30]. In a d d i t i o n , the in vitro p r o l i f e r a t i o n o f the THP1 m o n o b l a s t i c cell line is also a c c o m p a nied by m a t u r a t i o n in s o m e cell s u b p o p u l a t i o n s [7]. O n the o t h e r hand, we also have r e p o r t e d recently that cultured m a c r o p h a g e s secrete a factor which inhibits the m a t u r a t i o n o f p e r i p h e r a l b l o o d m o n o c y t e s [27]. Thus, it a p p e a r s t h a t und e r s t a n d i n g o f the m e c h a n i s m which regulates the b a l a n c e between p r o l i f e r a t i o n a n d differentiat i o n m a y be a crucial step in the u n d e r s t a n d i n g o f m a l i g n a n t processes. T h e finding o f s p o n t a n e ous d i f f e r e n t i a t i o n in p r i m a r y cultures o f A M M L - d e r i v e d cells a n d its possible i n h i b i t i o n by m a t u r e m a c r o p h a g e s m a y help to u n d e r s t a n d this m e c h a n i s m in the future.

References [1] Epstein, A. L. and Kaplan, H. S. (1974) Cancer 34, 1851. [2] Lozzio, C. B. and Lozzio, B. B. (1975) Blood 45, 321. [31 Sundstrom, C. and Nilsson, K. (1976) Int. J. Cancer 17, 565. [4] Collins, S. J., Gallo, R. C. and Gallagher, R. T. (1977) Nature (London) 270, 347. [5] DePersio, 3. E, Brennan, J. K., Lichtman, M. A. and Speiser, B. L. (1978) Blood 51, 507. [6] Koeffler, H. P. and Golde, D. W. (1978) Science 200, 1153. [7] Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T. and Taka, K. (1980) Int. J. Cancer 26, 171. [8] Ben-Bassat, H., Korkesh, A., Voss, R., Leizerowitz, R. and Polliack, A. (1982) Leukemia Res. 6, 743. [9] Collins, S. J., Ruscetti, F. W., Gallagher, R. E. and Gal1o, R. C. (1978) PNAS 75, 2458. [10] Lotem, J. and Sachs, L. (1979) PNAS 76, 5158. [11] Koeffler, H. P. (1983) Blood 62, 709. [12] Koren, H. S., Anderson, S. J. and Larrick, J. W. Nature (London) 279, 328-330. [13] Koeffler, H. P. and Golde, D. W. Blood 56, 344. [14] Svet-Moldavskaya, I. A., Arlin, Z., Svet-Moldavsky, G. J. et al. (1981) Cancer Res. 41, 4335. [15] Butler, R. H., Revoltella, R. P., Musiani, P. and Piantelli, M. (1983) Cell Immunol. 78, 368. [16] Treves, A. J., Halperin, H., Barak, V., Bar-Tana, R., Halimi, M., Fibach, E., Gamliel, H., Leizerowitz, R. and Polliack, A. (1985) Exp Hematol. 13, 281. [17] Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77. [18] Treves, A. J., Yagoda, D., Haimovitz, A., Ramu, N., Rachmilewitz, D. and Fuks, Z. (1980) J. Immunol. Methods 39, 71. 229

[19] Fibach, E., Peled, T., Treves, A., Kornberg, A. and Rachmilewitz, E. A. (1982) Leukemia Res. 6, 781. [20] Breitman, T. R., Selonick, S. E. and Collins, S. J. (1980) PNAS 77, 2936. [21] Roberts Jr., N. J., Douglas Jr., R. G., Simons, R. M. and Diamond, M. E. (1979) J. Immunol. 123, 365. [22] Broxmeyer, H. E., Bognacki, J., Ralph, P., D6rmer, M. H., Li, L. U. and Castro-Melaspina, H. (1982) Blood 60, 595 [23] Kurland, J. I. and Bockman, R. (1978) J. Exp. Med. 147, 952. [24] Broxmeyer, H. E., Lu, L., Platzer, E., Feit, C., Juliano, L. and Rubin, B. Y. (1983) J. Immunol. 131, 1300.

230

[25] Kurland, J. 1. and Moore, M. A. S. (1977) Exp. Hematol. 5, 357. [26] Pelus, L. M., Broxmeyer, H. E., K~drland, J. I. and Moore, M. A. S. (1979) J. Exp. Med. 150, 277. [27] Maoz, H., Polliack, A., Barak, V., Yatziv, S., Biran, S., Giloh, H. and Treves, A. J. (1985) Int. J. Cell Cloning, in press. [28] Jondal, M. and Klein, G. (1973) J. Exp. Med. 138, 1365. [29] Salahuddin, S. Z., Markham, R D., Ruscetti, E W. and Gallo, R. C. (1981) Blood 58, 931. [30] Salahuddin, S. Z., Markham, R D. and Gallo, R. C. (1982) J..Exp. Med. 155, 1842.