DEVELOPMENT OF AN IN VIVO MODEL OF HUMAN MULTIPLE

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Development of an In Vivo Model of Human Multiple Myeloma Bone Disease By Melissa Alsina, Brendan Boyce, Rowena D. Devlin, Judith L. Anderson, Fiona Craig, Gregory R. Mundy, and G. David Roodman Osteolyticbonedestructionand its complications, bone pain, pathologic fractures, and hypercalcemia, are a major source of morbidity and mortality in patients with multiple myeloma. The bone destruction in multiple myeloma is due t o increased osteoclast (OCL) activity and decreased bone formation in areas of bone adjacent t o myeloma cells. The mechanisms underlying osteolysis in multiple myeloma in vivo are unclear. We used a human plasma cell leukemia cell line, ARH-77, that has disseminated growth in mice with severe combined immunodeficiency (SCID) and expresses IgGK, as a model for human multiple myeloma. SCID mice were irradiated with 400 rads and micewere injected either with 10’ARH-77 cells intravenously (ARH-77 mice) or vehicle 24 hours after irradiation.Development of bone disease was assessed by bloodionized calcium levels, x-rays, and histology. All ARH-77, but none of control mice that survived irradiation, developed hind limb paralysis 28 t o 35 days after injection and developed hypercalcemia (1.35 t o 1.46 mmol/ L) a mean of 5 days after becoming paraplegic. Lytic bone lesions were detected using x-rays in all thehypercalcemic mice examined. No lytic lesions or hypercalcemia developed in the controls. Controls or ARH-77 mice, after developing hypercalcemia, were then killed and bone marrow plasma from the long bones was obtained, concentrated, and assayed for boneresorbingactivity. Bone marrow plasma from ARH-77 mice induced significant bone resorption in the fetal

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HE MECHANISMS responsible for the extensive bone destruction in multiple myeloma are not well understood due to lack of a good human cell model of multiple myeloma bone disease. Bone destruction is a prominent clinical feature of almost all patients with multiple myeloma. In addition to infections, it is responsible for many of the most debilitating clinical features of the disease, including intractable bone pain, fractures occurring either spontaneously or after trivial injury, and hypercalcemia with its attendant signs and symptoms.’ The bone disease in multiple myeloma is mainly osteolytic with increased osteoclastic bone resorption in areas of bone adjacent to myeloma cells.’ In addition, bone formation is also decreased in patients with high tumor burden^.^ These data suggest that locally acting factors produced by myeloma cells play an important role in the extensive bone destruction seen in these patients. To date, these factors have not been clearly identified in vivo. Therefore, as an initial step to clarify the mechanisms responsible for the osteolytic bone destruction in multiple myeloma, we developed an in vivo model of human myeloma bone disease that mimicked the disease in humans and could allow identification of the factors responsible for the intensive bone destruction in vivo based on the studies of Huang et al.4 They previously reported that the human myeloma cell line (AM-77) had disseminated growth in SCID mice and documented the presence of human IgG in the ARH-77 mice serum. We have used this cell line to successfully develop an in vivo model of human myeloma bone disease, as will be described in this report. MATERIALS AND METHODS Preparation of ARH-77 multiple myeloma cell line conditioned media. ARH-77 cells, an IgGK-secreting human plasma cell leukeBlood, Vol 87, No 4 (February 15). 1996: pp 1495-1501

rat long bone resorption assay when compared with controls (percentage of total ‘%a released = 35% ? 4% v 11% f 1%). Histologic examination of tissues from the ARH-77 mice showed infiltration of myeloma cells in the liver and spleen and marked infiltration in vertebrae and long bones, with loss of bony trabeculae and increased OCL numbers. Interestingly, cultures of ARH-77 mouse bone marrow for early OCL precursors (colony-formingunit-granulocytemacrophage [CFU-GM11showed a threefold increase in CFUGM from ARH-77 marrow versus controls (185 f 32 v 40 f 3 per 2 x I O 5 cells plated). Bone-resorbing human and murine cytokines such as interleukin-6 (IL-6). IL-la or p, TGFa, lymphotoxin, and TNFa were not significantly increased in ARH-77 mouse sera or marrow plasma, compared with control mice, although ARH-77 cells produce IL-6 and lymphotoxin in vitro. Conditioned media from ARH-77 cells induced significant bone resorption in the fetal rat longbone resorption assay when compared with untreated media (percentage of total 45Careleased = 22% ? 2% W 11% & 1%). This effect was not blocked by anti-IL-6 or antilymphotoxin (percentage of total “Ca released = 19% ? 1% and 22% & l%, respectively). Thus, we have developed a model of human multiple myeloma bone disease that should be very useful t o dissect the pathogenesis of the bone destruction in multiple myeloma. 0 1996 by The American Society of Hematology.

mia cell line, were generously provided by Dr E. Vittela (University of Texas Southwestern Medical Center, Dallas, TX). ARH-77 cells were plated at 2.5 X lo5 cells/mL in RPMI-1640 (GIBCO, Grand Island, NY) containing 20% fetal calf serum (FCS; Hyclone Laboratory, Logan, UT) and cultured for 5 days. Conditioned media from the cultures was collected and concentrated 4X using a Microconcentrator Centriprep 3 (Amicon, Danvers, MA). Transplantation of ARH-77cells in SCID mice. Female SCID mice (6 to 8 weeks old) were obtained from the University of Wisconsin Gnobiote Laboratory. Mice were irradiated with 400 rads using a @ C O ‘ source and 24 hours after irradiation were injected in the tail vein with lo6 ARH-77 cells (ARH-77 mice) intravenously. Mice that were irradiated but injected with vehicle rather than cells

From the Department of Medicine, Divisions of Hematology and Endocrinology, and the Department of Pathology,University of Texas Health Science Center at San Antonio and Audie Murphy Veterans Administration Medical Center, San Antonio, TX. Submitted July 5, 1995; accepted September 15, 1995. Supported by Research Funds from the Veterans Administration and Grant No. AM35188 from the National Institutes of Diabetes and Digestive and Kidney Disease; Grants No. CA-40035 and K12 Training Grant No. CA01723 from the National Cancer Institute; and Grants No. AR39529 and AR41336 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. Address reprint requests to G. David Roodman, MD, PhD, ResearcWHematology (151), Audie Murphy Veterans Administration Hospital, 7400 Merton Minter Blvd, San Antonio, TX 78284. The publication costsof this article were defrayedin part by page chargepayment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8704-0045$3.00/0 1495


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served as a control group. Mice were then followed-up weekly by measurement of serum calcium levels and whole body x-rays. When the ARH-77 mice became hypercalcemic (whole blood Ca++,> 1.35 mmol/L), they were anesthetized with methoxyflurane (Pitman Moore, Mundelein, IL) and killed by cervical dislocation. Marrow cells and marrow plasma were then isolated from long bones as described below. Vertebral bones were dissected free of surrounding tissue and used for bone histomorphometry studies. Collection of bone marrow plasma and assay of early osteoclast precursors (colony-forming unit-granulocyte-macrophage [CFUC M ) . Femurs were removed aseptically and dissected free of adhering tissue. The ends of the femurs were cut with a scalpel blade and the marrow was flushed with 5 mL of a-minimal essential containing 0.1 % (vollvol) penicillin-streptomycin medium ((U-") (GIBCO) using a 25-gauge needle. The cell suspension was centrifuged at 4OOg for IO minutes and bone marrow plasma was collected and concentrated 5X using a Microconcentrator (Amicon, Danvers, MA). Bone marrow cells (5 X 106/mL)were resuspended in a"Eh4 containing 15% FCS (Hyclone Laboratory) and incubated for 2 hours at 37°C in a humid atmosphere of 5% COz-air to remove cells adherent to plastic. The nonadherent bone marrow cells (i05/mL) were plated on 35-mm tissue culture dishes (Falcon, Lincoln Park, NJ) in 1 mL of 0.8% methylcellulose (MC; Aldrich CO,Milwaukee, WI),supplemented with 20% FCS, 1% bovine serum albumin (BSA; Sigma Chemical CO,St Louis, MO), and 1.25 ng/mL of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGMC S F Immunex CO,Seattle, WA) as the source of colony-stimulating activity. Each assay was performed in triplicate. Cultures were incubated at 37°C in a humid atmosphere of 5% COz-air for 7 days, at which time colonies (>40 cells) and clusters (>IO and <40 cells) were counted with an inverted microscope. Bone resorption assays. Timed-pregnant rats were injected with 250 pCi of "CaCI2 at day 18 of gestation. One day later, the rats were killed by cervical dislocation and the embryos were removed. The explanted radii and ulnae were cultured on circles of mixed ester membrane filters (0.45 pm; Whatman, Hillsboro, OR) on stainless steel grids in 0.5 mL of chemically defined medium (Sigma) supplemented with 1 mg/mL BSA (Sigma) and penicillin-streptomycin (50 U/mL and 50 mg/mL, respectively) in 5% CO2 in air at 37°C. as modified by Raisz and Niemann? The radii and ulnae were incubated for 24 hours in control media to allow for the removal of the exchangeable 45Cabefore transferring to equilibrated control or experimental media. Experimental media contained varying concentrations of either bone marrow plasma from ARH-77 mice or control mice, media conditioned by ARH-77 cells in vitro, or untreated culture media. Control or experimental media were then changed after 72 hours. The bone explants were incubated for a total of 5 days. Bone-resorbing activity was measured as the percentage of total 45Careleased from the bone into the media over the 5 days of incubation. Assay ofbone-resorbing cytokines. The human and murine cytokines that induce bone resorption (human IL-IP, human TGFa, PTHrP, human IL-6, human TNFP, murine E-6, murine TNFB, and murine & l a ) were measured in the peripheral blood sera and bone marrow plasma of ARH-77 or control mice and the ARH-77 cell conditioned media using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Endogen, Boston, MA). The lower limit of detection for cytokines in these assays was approximately 10 pg/mL. Processing of specimens for histology. Bones from all animals were fixed in 10% phosphate-buffered formalin for 24 to 48 hours, decalcified in 14% EDTA for 2 to 3 weeks, processed through graded alcohols, and embedded in paraffin wax.Serial sections (3-pm thick) of vertebral bodies were cut at various levels and stained with hematoxylin, eosin, orange G, and phloxine for histologic analysis. Con-

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Fig 1. Induction of hypwcalcemia in mice injected with ARH-77

cells. ARH-77 mice (01developed dgnlfkant hyporcakemia (1.43 2 0.12 mmol/L Y 1.15 f 0.25 for c o n t r o l mice) 28to 36 days after injection. Results represent the mean f SEM for seven determinations. Similar results were seen in thrw indepondent experiments. P = .00013

secutive sections (2-pm thick) were also taken at various levels to allow us to examine the same cell for expression of tartrate-resistant acid phosphatase (TRAP), a marker enzyme of osteoclasts. These sections were deparaffinized in xylene and immersed in acetone for 5 minutes. They were then placed for 1 hour in the substrate solution that contained 0.09 mmoVL naphthol-AS-B1 phosphate (Sigma) in 0.2 moyL acetate and 0.4 m o m L-(+)-tartaric acid (Sigma) at pH 4.9. Sections were then placed for 30 minutes in hexazotized pararosaniline in 0.2 moUL acetate buffer with 0.4 m o m tartaric acid, rinsed, and counterstained with methyl green and light green SF yellowish (Sigma). Staristical analysis. Results are expressed as the mean ? the standard error of the mean (SEM) and are presented for typical experiments. Results were similar in two or more independent experiments. Differences were compared using Student's t-test or ANOVA and were considered significant for P values <.05. RESULTS

ARH-77 mice. AllARH-77 mice thatsurvivedirradiation developed hind limb paralysis 28 to 35 days after the injection of the cells and lost 10% of their lean body mass bythe time they becomeparaplegic.Figure 1 shows the whole blood calciumlevels in ARH-77 or controlmice over the course of the experiments. All the ARH-77 mice developedhypercalcemiaapproximately 5 days after becoming paraplegic, with a mean whole blood ionized calcium of 1.43 mmoliL (range, 1.35 to 1.46 mmoliL). Multiple lytic lesions and diffuse osteopenia were detected in the hypercalcemic mice by x-rays (Fig2). Neither hypercalcemia nor lytic bone lesions developed in the controls (P = .0002). Cytokines. Levels of knownbone-resorbinghumanand murine cytokines were measured in the peripheralblood sera andbonemarrowplasma of ARH-77orcontrol mice, as well as inmediaconditionedbyARH-77 cells. Although ARH-77 cells secrete IL-6 (120 pg/mL) and TNFP (800 pg/ mL) in vitro, these cytokines were undetectable in the bone marrow plasma or sera from the ARH-77 mice. Human IL-


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Fig 2. Radiologic examination

of ARH-77 mice. Lytk lesions are present in ARH-77 mice through out axialskeleton(arrows).Lytic

10 was detected in the bonemarrow plasma of ARH-77 mice at a concentration of 20 pg/mL, a level incapable of stimulating bone resorption.” Murine IL-6 and 1L-l were detected in both the ARH-77 mice and control bone marrow plasma and serum, respectively, but the levels did not differ significantly. Effects of bone marrow plasma from ARH-77 mice and ARH-77 cells conditioned media on bone resorption. Bone marrow plasma from ARH-77 mice induced significant bone resorption in the fetal rat long bone resorption assay when compared with bone marrow plasma from controls (percentage of total 4sCa released = 35% ? 4% v 1 1% ? l%), as shown in Fig 3. Conditioned media from ARH-77 cells induced significant bone resorption in the same assay when compared withuntreatedmedia (percentage of total 4sCa released = 22% 2 2% v 11% 2 1 %), as shown in Fig 4. Antibodies against TNF and lymphotoxin failed to block this effect significantly (percentage of total 4sCareleased = 22% ? 1% and 19% ? l%, respectively; Fig 4).A similar pattern of bone resorption activity was seen in two independent experiments. Analysis of early osteoclast precursors in ARH-77 mice. To determine if osteoclast precursors were increased in marrow of ARH-77 mice compared with controls, cultures for early osteoclast precursors (CFU-GM) were performed. CFU-GM colonies were increased threefold in ARH-77 mice versus controls (185 t 32 v 40 2 3 per 2 X IO5cells plated; Fig 5 ) . Histology. The bonemarrow of the ARH-77 mice was infiltrated by ARH-77 cells (Fig 6). Immunostaining of the bone marrow cells for human K and A light chains showed plasmablasts that expressed K light chains and not A light chains. Histologically, the ARH-77 mice showed infiltration of myeloma cells in the liver, spleen, and bones. The verte-

brae and long bones had marked infiltration by the tumor with loss of bony trabeculae and increased osteoclast numbers without an osteoblastic response (Fig 7A). Deep resorption pits were associated with the osteoclasts adjacent to myeloma cells. In contrast, the bone next to normal bone marrow had a smooth contour. This increase in osteoclast numbers was even more dramatic when the bone sections were stained for TRAP, a marker enzyme for osteoclasts (Fig 7B). There was a marked increase in osteoclast numbers

Control Bone Marrow Plasma

ARH-77 Bone Marrow Plasma

Fig 3. Effects of ARH-77 mouse bone marrow plasmaon bone resorption in the fetal rat long boneassay.Bone marrow plasma from ARH-77 mice induced significant bone resorption in the fetal rat long bone resorption assay when compared with control mouse bone marrow plasma (percentageof total “Ca released = 35% 2 4% Y 11% ? 1%; P = ,027). Results represent the mean 2 SEM for four determinations.Similar results were seen in two independentexperiments.


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Untreated Media

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in areas of bone adjacent to myeloma cells, but not in areas of bone adjacent to normal bone marrow. Human K and A light chain immunostaining. Cytospin slides were prepared with IO' ARH-77 cells/slide and dried overnight. The slides were then fixed with acetone, dried, and rehydrated with phosphate-buffered saline (PBS). After suspending the slides in blocking solution of 10% ovalbumin, the peroxidase conjugated rabbit antihuman A light or rabbit human K light chain (Dako, Carpinteria, CA) were applied. Aminoethylcarbizide chromogen was applied as substrate, and the slides were then counterstained with Biomedia hematoxylin. Cells positive for K or A light chains showed intense red staining of the cytoplasm.

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Fig 5. Quantitation of early osteoclast precursors in marrow cultures from ARH-77 mice and controls. Early osteoclast precursors were significantly increasedin ARH-77 mouse bone marrow cultures when compared with controls (CFU-GM colonies/2 x 10' cells plated = 185 f 32 v 40 f 3; P = .011). Results representthe mean f SEM for three determinations. Similar results were seen in twoindependent experiments.

Fig 4. Effects of ARIH-77 cell conditioned media on bone resorptionin fetal rat long bone assay. ARH77 cell conditioned media induced significant bone resorptionin the fetal rat long bone resorption assay when compared with untreated media (percentage of total "Ca released = 22Y0 f 2% v 11% f 196; P = .0000551. Treatment with anti-TNFp at 1 p g / m L which can neutralize 1 nglmL TNFP, and anti-11-6 at 0.2 pg/mL, which can neutralize 1 ng/mL 11-6, failed to block bone resorption induced bythe ARH77 cell conditioned media (percentage of total %a released = 22% f 1% and 19% f 1%. respectively: P = .220). Results are presented as the mean f SEM for four determinations.Similar resultswere seen in t w o independent experiments.

DISCUSSION

Bone destruction is one of the most prominent features of multiple myeloma and is present in about 80% of patients.' The precise molecular mechanisms responsible for the bone destruction in multiple myeloma remain unclear, but observations over time have shown a number of facts. The mechanism by which bone is destroyed inmyelomaisviathe osteoclast, the normal bone-resorbing cell.' Osteoclasts accumulate on bone-resorbing surfaces in myeloma only adjacent to collections of myeloma cells and not in areas adjacent to normal bone marrow. Thus, it appears that the mechanism by which osteoclasts are stimulated in myeloma is a local one. In addition, bone formation isinhibitedwhentumor burden is high, resulting in uncoupling of normal bone remodeling? It is therefore likely that interactions between myeloma cells and bone cells play an important role in the development of bone disease. Myeloma cells may produce factors that affect osteoclasts, osteoclast precursors, and/or osteoblasts, thereby uncoupling normalboneremodeling. Osteoblasts may be stimulated to produce factors that enhance osteoclastic bone resorption, andor osteoclasts themselves may produce factors that may stimulate the growth of myeloma cells, acting to amplify the effects of myeloma on bone resorption. Since the initial description of a myeloma-derived osteoclast activating factor in 1974 byMundy et al," several cytokines have been identified that are produced by human myeloma cell lines and induce bone resorption in fetal rat long bone resorption assays in vitro, including IL-I, IL-6, lymphotoxin, and transforming growth factor Among these, a leading candidate for the cytokine responsihle for the bone destruction associated with myeloma is IL-6. IL-6 has been shown to be a myeloma growth factor in vivo14.1s and is known to stimulate osteoclastic bone resorption and osteoclast formation," and osteoblasts, which have been reported to be recruited to areas of bone marrow involvement in early multiple myeloma, secrete IL-6." Nev-


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Fig 6. Bona marrow involvamant by ARH-77 cellsin vivo. (A) Wright-Giemsa stain of ARH-77 mouse bona marrow cells showing plasmoblastswith basophilic cytoplasm, high nuclear-cytoplasm ratio, and prominent nucleoli. (B) Human K light chain immunostaining of bone marrow cells from ARH-77 mica. Plasmoblasts stained positively for cytoplasmic K light chains.

ertheless, IL-6 has not beenfound to be consistently elevated in sera of patients with multiple myeloma and the level of IL-6 has notbeen correlated with the extent of multiple myeloma bone disease. Furthermore, IL-6 is a weak boneresorbing factor.I8 Therefore, the role that IL-6 plays in the pathogenesis of myeloma bone disease in vivo is not clearly understood. In addition, there maybe other cytokines that are active in myeloma bone disease, but, to date, they have not been clearly identified. To help clarify the mechanisms of bone destruction in multiple myeloma, it is necessary to develop an in vivo model of human myeloma bone disease that mimics the bone

disease in humans and allows identification of the factors playing a role in the bone destruction in vivo. Rad1 et al'9.2" reported that aging C57BWKalwRij mice developed spontaneous multiple myeloma with bone marrow involvement and diffuse osteolytic bone lesions. Transplantation of the bone marrow cells of these animals to youngeranimals of the same strain successfully induced disseminated multiple myeloma. Even though the disease in these animals resembles that in humans in many aspects, including the presence of osteolytic bone destruction, its usefulness to study the pathogenetic mechanisms of multiple myeloma bone disease in humans is limited by the fact that it represents a modelof mouse


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Fig 7. ARH? -l celk induced osteodastosis and mnrked bone resorption in vivo. (A) shows l W x magnification of H&€ stain of vertebral bone section from ARH-77 mice. Note that osteoclast n u m bers (arrows) are markedly increased and associated with deep resorption pits in areas of bone adjacent to myeloma (MM), whereas they are not increased adjacentto areas of normal bone marrow (NBM). (B)shows a TRAP stain of ARH-77 mice bone section. Osteoclasts stain intenselyfor TRAP activity (arrows).

multiple myeloma and that the cytokines involved may not necessarily represent those involved in human multiple myeloma. For example, IL-6 is a potent stimulator of human butnot murine osteoclast formation andbone resorption. Therefore, other investigators have tried to develop animal models of multiple myeloma using human myeloma cells. Feo-Zuppardi et al" reportedlong-term engraftment of freshly isolated primary human myeloma cells after intraperitoneal injection in SCID mice. Even though they documented circulating levels of human IgG in these mice for more than 30 days after injection of the cells, these mice didnot develop disseminated disease or bonemarrow involvement, but only had collections of plasma cells in the peritoneal cavity.

Other investigators have also used the intraperitoneal route to inject a murine plasmacytoma into Balb/c mice as a model for human multiple myeloma,22and again the mice failed to develop the disseminated disease or bone involvement that characterizes human multiple myeloma. Recently, Huang et ala successfully transplantedand showed engraftment of a human multiple myeloma cell line, ARH-77, in SCID mice. The mice developed disseminated disease withbonemarrow involvement and some microscopic osteolytic lesions in the vertebrae and bones of the skull. Because these cells showed disseminated growth in SCID mice, we have used transplantation of ARH-77 cells into SCID mice to develop an in vivo model ofhuman myeloma bone disease. This model is unique in that all the


IN VIVO MODEL OF HUMAN MYELOMA BONEDISEASE

mice injected with the human myeloma cell line developed hypercalcemia and osteolytic bone lesions, as assessed radiographically. Histologically, the ARH-77 mice developed osteolytic bone destruction with increased osteoclastic bone resorption in areas of bone adjacent to myeloma cells but not in areas of bone adjacent to normal bone marrow. To determine if early osteoclast precursors were increased in ARH-77 mice as well, we performed C m - G M cultures from the bone marrow cells of the ARH-77 mice and cont r o l ~ . *The ~ bulk of recent evidence supports CFU-GM as the earliest identifiable osteoclast precursor.'7.23.24 CFU-GM colony formation was significantly elevated in the AM-77 mice as compared with the control mice, suggesting that the myeloma cells induce not only mature osteoclast recruitment to the areas of bone adjacent to the tumor, but also new osteoclast formation. Furthermore, bone marrow plasma from these ARH-77 mice and conditioned media from ARH-77 cells induced bone resorption in the fetal rat long bone resorption assay. Levels of either human or murine cytokines known to induce bone resorption were not significantly elevated in the bone marrow plasma or serum of these mice, although ARH-77 cells produce significant amounts of L-6 and TIWP in vitro. Furthermore, neutralizing antibodies against L-6 and lymphotoxin failed to block the bone resorption induced by ARH-77 conditioned media in the fetal rat long bone resorption assay. These data suggest that other factors produced by myeloma cells or the marrow microenvironment may play an important role in the bone disease in multiple myeloma. This model should be very helpful in the identification and characterization of these factors, which may lead to novel therapeutic strategies that improve the quality of life and survival of these patients. ACKNOWLEDGMENT

The authors thank Bibi Cates for excellent preparation of this manuscript. REFERENCES 1. Mundy GR, Bertolini D R Bone destruction and hypercalcemia in plasma cell myeloma. Semin Oncol 3:291, 1986 2. Bataille R, Chappard D, Klein B: Mechanisms of bone lesions in multiple myeloma. Hematol Oncol Clin North Am 6:285, 1992 3. Taube T, Beneton MN, McCloskey EV, Rogers S,Greaves M, Kanis JA: Abnormal bone remodelling in patients with myelomatosis and normal biochemical indices of bone resorption. Eur J Haematol 49: 192, 1992 4. Huang W ,Richardson JA, Tong AW, Zhang BQ, Stone MJ, Vitetta ES: Disseminated growth of a human multiple myeloma cell line in mice with severe combined immunodeficiency disease. Cancer Res 53:1392, 1993 5. Raisz LC, Neimann I: Effect of phosphate, calcium and magnesium on bone resorption and hormonal responses in tissue culture. Endocrinology 85:446, 1969 6. Gowen M, Meikle MC, Reynolds JJ: Stimulation of bone re-

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sorption in vitro by anon-prostanoid factor released by human monocytes in culture. Biochim Biophys Acta 762471, 1983 7. MacLennan IC,Drayson M, Dunn J: Multiple myeloma (review). Br Med J 308:1033, 1994 8. Bataille R, Chappard D, Alexandre C, Sany J: Importance of quantitative histology of bone changes in monoclonal gammopathy. Br J Cancer 53:805, 1986 9. Bataille R, Chappard D, Marcelli C, Dessauw P, Sany J, Baldet P, Alexandre C: Mechanism of bone destruction in multiple myeloma. The importance of an unbalanced process in determining the severity of lytic bone disease. J Clin Oncol 7:1909, 1989 10. Mundy GR, Raisz LC, Cooper RA, Schechter GP, Salmon S E Evidence for the secretion of an osteoclast stimulating factor in myeloma. N Engl J Med 291:1041, 1974 11. Kawano M, Tanaka H: Interleukin-l accelerates autocrine growth of myeloma cells through interleukin-6 in human myeloma. Blood 73:2145, 1989 12. Garrett IR, Durie BGM, Nedwin GE, Gillespie A, Bringman T, Sabatini M, Bertolini DR. Mundy G R Production of the bone resorbing cytokine lymphotoxin by cultured human myeloma cells. N Engl J Med 317:526, 1987 13. Cozzolino F, Torua M: Production of interleukin-l by bone marrow myeloma cells. Blood 74:380, 1989 14. Klein B, Bataille R Cytokine network inhuman multiple myeloma. Hematol Oncol Clin North Am 6:273, 1992 15. Bataille R, Jourdan M, Zhang XG, Klein B: Serum levels of interleukin-6, a potent myeloma cell growth factor, as a reflection of disease severity in plasma cell dyscrasias. J Clin Invest 84:2008, 1989 16. Kurihara N. Bertolini D, Suda T, Akiyama Y,Roodman CD: IL-6 stimulates osteoclast-like multinucleated cell formation in longterm human marrow cultures by inducing IL-l release. J Immunol 144:4226, 1990 17. De La Mata J, Uy HL, Guise TA, Stoly B, Boyce BF, Mundy GF, Roodman CD: IL-6 enhances hypercalcemia and bone resorption mediated by PTH-rP in vivo. J Clin Invest 95:2846, 1995 18. Linkhart TA, Linkhart SG, Strong DD: Interleukind messenger RNA expression and interleukin-6 protein secretion in normal human osteoblast-like cells: Regulation by interleukin-l. J Bone Miner Res 6:1285, 1991 19. Croese J W , Vas Nunes CM, Rad1 J, van den Enden-Vieveen MH, Brondijk RI, Boersma WJ: The 5T2 mouse multiple myeloma model: Characterization of 5T2 cells within the bone marrow. Br J Cancer 56:555, 1987 20. Rad1 J, Croese JW, Zurcher C, van den Enden-Vieveen MH, de k u w AM: Animal model of human disease. Multiple myeloma. Am J Pathol 132593, 1988 21. Feo-Zuppardi FJ, Taylor CW, Iwato K, Lopez MHA, Grogan TM, Odeleye A, Hersh EM, Salmon S E Long-term engraftment of fresh human myeloma cells in SCID mice. Blood 802843, 1192 22. Valeriote F, Grates H: MOR-315 murine plasmacytoma as a model anticancer screen for human multiple myeloma. J Natl Cancer Inst 76:61, 1986 23. Uy HL, Dallas M, Calland J W , Boyce BF, Mundy GR, Roodman CD: Use of an in vivo model to determine the effects of interleukin-l on cells at different stages in the osteoclast lineage. J Bone Miner Res 10:295, 1995 24. Kurihara N. Chenu C. Miller M, Civin CI, Roodman CD: Identification of committed mononuclear precursors for osteoclastlike cells formed in long-term marrow cultures. Endocrinology 126:2733, 1990


1996 87: 1495-1501

Development of an in vivo model of human multiple myeloma bone disease M Alsina, B Boyce, RD Devlin, JL Anderson, F Craig, GR Mundy and GD Roodman

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DEVELOPMENT OF AN IN VIVO MODEL OF HUMAN MULTIPLE

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