Chemotherapeutic Susceptibility of Human Bone Marrow Progenitor Cells and Human Myelogenous Leukemia Cells (HMO) in Co-Culture: Preliminary Report

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1 International Journal of Cell Cloning 2: (1984) Chemotherapeutic Susceptibility of Human Bone Marrow Progenitor Cells and Human Myelogenous Leukemia Cells (HMO) in Co-Culture: Preliminary Report Tatsumi Himori, Takao Ohnuma, James F. Holland Department of Neoplastic Diseases, Mount Sinai School of Medicine. New York, NY, USA Key Words. Co-culture. CFU-C. HL-60 Abstract. We determined the chemotherapeutic susceptibility of normal human granulocyte progenitor cells (CFU-C) and acute myelogenous leukemia cells (HL-60) in co-culture. Nucleated bone marrow cells and HL-60 cells were mixed in 0.3% agar containing McCoy s 5A medium, fetal bovine serum, human placenta-conditioned medium, and various concentrations of chemotherapeutic agents. They were incubated in 5% humidified COz at 37 C for 8-10 days. CFU-C and HL-60 colonies were differentiated morphologically. The formation of CFU-C was progressively inhibited with the increasing number of HL-60 cells, whereas the presence or absence of bone marrow cells did not influence the number of HL-60 colonies. In separate culture, HL-60 cells were more sensitive to vincristine than were CFU-C cells. In co-culture, however, the CFU-C became more sensitive to vincristine than in the separate culture. Similarly, co-culture CFU-C were more sensitive to daunorubicin than in separate culture. These data indicate that HL-60 leukemic cells exert inhibitory effects on normal bone marrow CFU-C; in such an inhibited state, normal bone marrow is more susceptible to certain chemotherapeutic agents. Introduction One of the most characteristic features of acute myelogenous leukemia (AML) is the development of profound suppression of normal hematopoiesis leading to serious complications from neutropenia, thrombocytopenia, and/or anemia. Correspondence: Dr. Takao Ohnuma, Department of Neoplastic Diseases, Mount Sinai School of Medicine, New York, NY (USA). Received December 15, 1983; accepted March 22, /84/$2.00/ AlphaMed Press, Inc.

2 Himori/Ohnuma/Holland 255 Co-culture in vitro of normal bone marrow cells and cells from patients with leukemia revealed that the latter inhibited normal bone marrow granulocyte-macrophage progenitor colony-forming cells (CFU-C) [ This effect was shown to be due to one or more humoral factors derived from leukemic cells [4-8]. A human AML cell line (HL-60) [91 was reported to produce a leukemia-associated inhibitor which was biochemically indistinguishable from that of AML or chronic myelogenous leukemia (CML) cells [ 101. Since chemotherapeutic efficacy is often expressed by the therapeutic index (i.e., cytotoxic effects on tumor cells versus toxicity to the host tissues), it is important to compare the effects of chemotherapeutic agents on target tissue with those on chemotherapeutically vulnerable normal tissue. In vitro comparisons of the inhibitory effects of chemotherapeutic agents on human leukemic cells and normal hematopoietic stem cells have been carried out [ 111. However, these comparisons for human CFU-C and tumor cell colonies were done using separate dishes. We determined the chemotherapeutic susceptibility of normal CFU-C and established human AML cells in co-culture. Materials and Methods Cells Bone marrow specimens were obtained from four healthy volunteers and five patients with head and neck cancer who had no bone marrow involvement and no prior chemotherapy and/or radiotherapy. The bone marrow was aspirated into a syringe previously made wet with heparin (liqueamin sodium, Organon, Inc., West Orange, NJ), immediately brought to the laboratory, and centrifuged at 700 g for 10 min. The buffy coat was separated using a pipette, washed twice with serum-free McCoy s 5A medium (GIBCO Laboratories, Grand Island, NY), and resuspended in the same medium with a final concentration of 2-4 x 106 cells/ml. HL-60 cells were obtained from Dr. J. Minowada (Roswell Park Memorial Institute, Buffalo, NY) and maintained in RPMI 1640 medium (GIBCO) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, GIBCO). Drugs Chemotherapeutic agents used were vincristine (VCR; Eli Lilly, Indianapolis, IN) and daunorubicin (DNR; obtained through the National Cancer Institute, Bethesda, MD). The drug solutions were freshly prepared with Dulbecco s phosphate-buffered saline (GIBCO) for each experiment. Experimental Procedures To study the suppressive effects of HL-60 cells on normal marrow CFU-C, we mixed 105 nucleated marrow cells and HL-60 cells ranging from 102 to 104 in

3 Human CFU-C and AML Cells in Co-Culture % (final concentration) agar containing McCoy s 5A supplemented medium [12], 10% FBS, and 0.1 ml of human placenta-conditioned medium. The conditioned medium was prepared according to the procedure described by Burgess et al. [13, 141 and stored at -2OOC until use. For the study of chemotherapeutic susceptibility of co-cultured normal CFU-C and HL-60 cells, 2 x lo5 marrow cells and 5 X 103 HL-60 cells (which produce approximately 50% inhibition of the number of CFU-C colonies) were used. They were mixed in 0.3% (final concentration) agar containing McCoy s 5A supplemented medium, 10% FBS, 0.1 ml of human placenta-conditioned medium, and varying concentrations of chemotherapeutic agents to a total of 1.1 ml and plated in 35 mm Petri dishes (No. 5217, Lux Scientific, Newbury Park, CA). The cells were incubated in a humidified atmosphere containing 5% COz in air at 37 C for 8-10 days with continuous exposure to chemotherapeutic agents. The CFU-C and HL-60 colonies were enumerated using an inverted microscope at x 100 magnification. The CFU-C colonies consisted of smaller and transparent cells. They formed three types of colonies: compact, mixed, and dispersed. Morphologically, HL-60 colonies were clearly different from CFU-C colonies. They consisted of larger, opaque cells containing various amounts of granular (or highly light-reflective) material in the cytoplasm. The HL-60 cells gave rise to only clump-like colonies. Cell aggregation of more than 40 cells was defined as a colony for both CFU-C and HL-60 [ 151. Visual differentiation between CFU-C and leukemic colonies was not difficult. In order to make correct identification of the cell types, however, a morphological differentiation between CFU-C and HL-60 colonies was made in most of the dishes after Wright staining using the method described by Salmon and Buick [ 161. All experiments were carried out in triplicate. Probability values were calculated using the Student s t test. Results In the absence of chemotherapeutic agents, the formation of CFU-C colonies was inhibited by the increasing number of HL-60 cells (Fig. 1). In contrast, colony formation of HL-60 cells was not inhibited in the presence of normal bone marrow cells up to 2 X 105 cells/dish. Figure 1 illustrates the number of colonies formed from normal bone marrow nucleated cells in co-culture with HL-60 cells. In the presence of 5 x 103 HL-60 cells/plate, the number of CFU-C colonies in co-culture decreased to approximately 50% of those in control plates. In separate cultures the number of CFU-C per plate ranged with a mean of 61, whereas in co-culture with HL-60, the mean number of CFU-C per plate was 31 with a range of The number of CFU-C was sufficient to determine quantitatively the effects of chemotherapeutic agents to approximately LDsO.

4 Himori/Ohnuma/Holland 251 NUMBER OF CFU-C c MBER OFHL-6 Fig. 1. Colony formation of normal bone marrow CFU-C and HL-60 cells in co-culture. Bar = SD; NMC i= nucleated marrow cells. In a separate culture, HL-60 cells were more susceptible to VCR than CFU-C in all three marrow specimens tested. In co-culture, however, CFU-C became more susceptible to VCR. The dose-response curve of HL-60 to VCR was not influenced by the presence of CFU-C. Thus, in coculture the dose-response curve of CFU-C from two marrow samples moved to the left of that of HL-60. (Results from one of these marrow samples are shown in Figure 2.) The dose-response curve of CFU-C from the third specimen overlapped that of HL-60 in co-culture. In a single bone marrow sample, HL-60 and CFU-C were equally sensitive to DNR when cultured and assayed separately, and the sensitivity of HL-60 was not influenced by the presence of CFU-C. CFU-C, however, became more sensitive to DNR in the presence of HL-60 (Fig. 3).

5 Human CFU-C and AML Cells in Co-Culture 258 Fig. 2. Effects of vincristine on separate or co-cultured CFU-C (0) from donor No. 1 and HL-60 (0) cells. Dose-response curves were drawn using the number of colonies/dish with no drug added as a unit for both separate culture and co-culture. * = Significant differences (p < 0.05) of the surviving fractions between CFU-C and HL-60 colonies; bar = SD.

6 Himori/Ohnuma/Holland 259 Fig. 3. Effects of daunorubicin on separate or co-cultured CFU-C (0) from donor No. 2 and HL-60 (0) cells. * = Significant differences (p < 0.05) of the surviving fractions between CFU-C and HL-60 colonies; bar = SD.

7 Human CFU-C and AML Cells in Co-Culture 260 Discussion By growing normal human CFU-C and HL-60 colonies in the same dish, we confirmed that the leukemia cells exerted inhibitory effects on the CFU-C, but the CFU-C had no effect on the HL-60. When the co-culture of normal human marrow cells and leukemic cells was carried out in the past, leukemic cells did not give rise to colonies, and only CFU-C colonies were enumerated [l-31. By growing CFU-C colonies and colonies from a leukemic cell line in the same dish simultaneously, we were able to compare the chemotherapeutic susceptibility of the two types of colonies sideby-side. Regarding the mechanism of CFU-C suppression, leukemic cellmediated humoral factors were identified [l, 5, 71. However, their inhibitory properties were not identical and their structures have not yet been identified. While normal human marrow CFU-C and autochthonous leukemic cells have not been co-cultured, the murine system proved that leukemic cells were also inhibitory on syngeneic CFU-C [ 171. Whatever the mechanism of CFU-C inhibition involved, we observed that while CFU-C is equally or less sensitive than the leukemic cells in separate culture, CFU-C in co-culture became more susceptible to VCR and DNR. The reason CFU-C became more vulnerable in the presence of HL-60 is unclear. Follow-up observations after 8-10 days did not show any further increase in the number of CFU-C colonies, indicating that the process is not simply a slowing of colony formation. We used bone marrow specimens from healthy donors and from patients with previously untreated head and neck cancer who had no bone marrow involvement. Although the number of bone marrow specimens studied was small in each group of donors, there was no difference in the number of CFU-C produced and the degree of inhibition produced by HL-60. No trend was recognized in the drug-induced inhibition of the CFU-C colonies among the two groups. Whether this increased chemotherapeutic susceptibility of normal CFU-C in co-culture with leukemic cells can be generalized in other antileukemic agents remains to be studied. Nevertheless, our preliminary data clearly indicate that normal human granulocyte progenitor cells are inhibited due to co-existing leukemic cells, and in such an inhibited state may become more vulnerable to certain chemotherapeutic agents. We believe the methodology described in this paper and an extension of this study to a dormant pluripotent hematopoietic stem cell, e.g.,

8 Himori/Ohnuma/Holland 26 1 CFU-GEMM [ 181, would provide a means of better understanding the interaction between the normal marrow element and leukemic cells under chemotherapeutic intervention. Acknowledgments This work was supported by United States Public Health Service grants, CA and CA-15936, from the National Cancer Institute; by the Chemotherapy Foundation, Inc., New York, NY; by the T.J. Martell Memorial Fund, New York, NY; and by the United Leukemia Fund, Inc., Flushing, NY. References Chiyoda, S.; Mizoguchi, H.; Asano, S.; Takaku, F.; Miura, Y.: Influence of leukemic cells on the colony formation of human bone marrow cells in vitro. 11. Suppressive effects of leukemic cell extracts. Br J Cancer 33: (1976). Chiyoda, S.; Mizoguchi, H.; Kosaka, K.; Takaku, F.; Miura, Y.: Influence of leukemic cells on the colony formation of human bone marrow cells in vitro. Br J Cancer 31: (1975). Moms, T.C.M.; McNeil, T.A.; Bridges, J.M.: Inhibition of normal human in vitro colony forming cells by cells from leukemic patients. Br J Cancer 31: (1975). Broxmeyer, H.E.; Grossbard, E.; Jacobson, N.; Moore, M.A.S.: Evidence for a proliferative advantage of human leukemic colony forming cells in vitro. J Natl Cancer Inst 60: (1978). Broxmeyer, H.E.; Jacobson, N.; Kurland, J.; Mendelsohn, N.; Moore, M.A.S.: In vitro suppression of normal granulocytic stem cells by inhibitory activity derived from human leukemia cells. J Natl Cancer Inst 60: (1978). Olofsson, T.; Cline, M.J.: Inhibitor of hematopoietic cell proliferation derived from a human leukemic cell line. Blood 52: (1978). Olofsson, T.; Olsson, I.: Suppression of normal ganulopoiesis in vitro by a leukemia-associated inhibitor (LAI) of acute and chronic leukemia. Blood 55: (1980). Olofsson, T.; Olsson, I.: Biochemical characterization of a leukemia-associated inhibitor (LAI) suppressing normal granulopoiesis in vitro. Blood 55: (1980). Collins, S.J.; Gallo, R.C.; Gallagher, R.E.: Continuous growth and differentiation of human myeloid leukemic cells in suspension culture. Nature 270: (1977). Olofsson, T.; Olsson, I.: Suppression of normal granulopoiesis in vitro by a leukemia associated inhibitor (LAI). Exp Hematol8: suppl. 7, p. 68 (1980).

9 Human CFU-C and AML Cells in Co-Culture Oladipupo Williams, C.K.; Ohnuma, T.; Holland, J.F.: Inhibition by chemotherapeutic agents of human bone marrow progenitor cells and clonogenic cells of a lymphoblastic cell line. Eur J Cancer 17: (1981). 12 Pike, B.L.; Robinson, W.A.: Human bone marrow colony growth in agar gel. J Cell Physiol 76: (1970). 13 Burgess, A.W.; Wilson, E.M.A.; Metcalf, D.: Stimulation by human placental conditioned medium of hemopoietic colony formation by human marrow cells. Blood 49: (1977). 14 Nicola, N.A.; Metcalf, D.; Johnson, G.R.; Burgess, A.W.: Preparation of colony-stimulating factors from human placental conditioned medium. Leuk Res 2: (1978). 15 Moore, M.A.S.; Burgess, A.W.; Metcalf, D.; McCulloch, E.A.; Robinson, W.A.; Dicke, K.A.; Chervenick, P.A.; Bull, J.M.; Wu, A.M.; Stanley, E.R.; Goldman, J.; Testa, N.: Report of a workshop on standardization of selective cultures from normal and leukemic cells. Br J Cancer 35: (1975). 16 Salmon, S.E.; Buick, R.M.: Preparation of permanent slides of intact softagar colony cultures of hematopoietic and tumor stem cells. Cancer Res 39: (1979). 17 Quesenberry, P.J.; Rappaport, J.M.; Fountebuoni, A.; Sullivan, R.; Zuckerman, K.; Ryan, M.: Inhibition of normal murine hematopoiesis by leukemic cells. N Engl J Med 299: (1978). 18 Fauser, A.A.; Messner, H.A.: Proliferative state of human plunpotent hematopoietic progenitors (CFU-GEMM) in normal individuals and under regenerative conditions after bone marrow transplantation. Blood 54: (1979).