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Phenotypic characterisation of mononuclear and multinucleated cells of giant cell tumour of bone
BonewdMinml.
16(1992)37-48
37
Efsevier BAM
w417
Phenotypic characterisatiou of mononuclear and multinucleated cells of giant cell tumour of bone
C.J. Joyner’, J.M. Quint#, J.T. Triffitt’, M.E. Oven’ and N.A. Athanasou* ‘MRC Bone Researchioboromy. Nuffifd Orthopedic Cmm. Ozfwd and *Nufield Lkpmmm Paholo#y. University of Oxford, John Radcliffe Ho~pml. Oxfonl, UK
of
(Received 18April 1991) (Awepled
lOSeptember 1’9%)
The giant cell tumor- of bone (GCIB) is a relatively uncommon primary bone tumottrwhich usually produces an expansile lytic lesion in the epiphysis and metaphy-
sis of 8 long bone [l?,]. Histo,ogically, these turnours are distinctive, coasisdog of an ovoid or spindle shaped mononuclear stromal cell component amongst which are scattered osteoclast-like multinucleated giant cells. There has been considerable
38
controversy surrounding the nature of the two cellular components. In particular, the identity of the cell type(s) forming the mononuclear cell component, the relationshia of the mononuclear stromal cells to the osteoclast-like giant cells and the nature of the proliferating cell in GCfB have not been clarified. The giant cells have numerous ultrastructural [3-S], cytochcmical [S-B], immunohistochemical [911] and functional [i2] features in common with the osteoclast. The mononuclear cell component is heterogeneous and composed of macrophages, fibroblast-like stromal cells and cells intermediate between these two types [3,4,13-151. In this study, we have sought to define further the nature of the cell types present in both the mononuclear and giant cell components of GCI’B at the time of resection and following culture in vitro and as subcutaneous implant in athymic mice. Cells from 3 cases of human GClB have been analysed in terms of their morphology, antigenic phenotype, cytochemistry and functional characteristics. These features have been compared with those of macrophages, osteoclasts and osteoblasts in order to establish whether there is a possibkcrelationship between these cells and the mor.o- or multinucleated cells of the GCTB.
Materials and Methods
Three GCTBs were examined in this study. Two were removed from the proximal femur, and one from the proximal humerus of patients aged 21, SOand 22 years respectively. Samples of the tumour were snap-frozen and stared in liquid nitrogen for cryostat sectioning; samples were also fued in formalin for routine histological processing and staining to confirm the diagnosis of GCTB. The rest of the tumour was either used fresh for functional studies, in vitro culture and in vivo culture as subcutaneous implant or frozen down for subsequentUSCin each of these types of study. Tissue for freezing was first cut int;, tiny pieces (2 nuns) then incubated in FCS (Gibw) containing 10% diiethylsulphoxide (DMSO) {IS min; UT) and finally cooled to -WC using a programmable ce.Ufreezer (Planer, Middlesex, UK). Samples were stored at -13YC until required for culture. On removal, the tumour was rinsed repeatedly in serum-free medium to remove the DMSO. of bone resorption Fresh pieces of tumour tissue were placed in RPM1 1640 tissue culture medium (Gibco) supplemented with antibiotics and 10% fetal calf strum (RPMUFCS). Portions of the tumour were then placed in a 35 mm Petri dish (Sterilin) and finely curetted with a scalpel blade.. The tissue suspension was then agitated with a glass pipette, heavier pieces allowed to settle and the suspension then pipetted into 16 mm wells of a Costar multiwell plate which contained 15 mm glass coverslips or cortical bone slices prepared as previously described [14. The cell suspension was settled on bone slices or coverslips for 30 min and then washed in RMFW’PCSand placed in fresh wells containing tissue culture medium. Cells cultured on bone slices and coverslips were incubated for up to 5 days, being removed after 24 h, 3 days and 5 days,
Response to calcitotdn and evidence
39 Table 1 Mottoclonal antibodies used in this study and immunoreactivity of ttmtott~lear cells (MO) and multinucleated giant cells (GCs) in original turnours and subctttaneous implants
PC10
I1
v9
1191
KPI PD7a6
IN
CR3143
Proliferaling cell nuclear antigen vimentin infcmlcdias fiaments CD68
+
WI
CD45(kVc-xy?C unnmonantipn)
1221
HLADR’
+
++
++
+
++
+
i+
at which time one of a pair of bone slices was fixed in 5% glutaraklehyde; the other was treated with Ttiton X-100 (0.1% in distilled water) to remove the cells from the bone surface and enable the full extent of resorption pit formation to be observed by scanning electron microcopy. Bone slices were dehydrated through a graded ethanol series, critical point dried from CO*, sputter coated with gold then examined in a Philips SEM 505 scatuting electron microscope. One of a pair of glass coverslips containing cell suspension was placed immediately after washing in tissue culture medium containing 1 &ml salmon calcitonin (APbammcmticals, Eastboume). The cakitonbt response ofmultinucleatcd and moaonuckar cells was observed continuously for up to 1 h and the response compared with cells on coverslips incubated in tissue culture medium alone. Cell suspensions ozt covetslips were fixed bt fotmalii after 1 h and stained with Giemsa. Cell suspensioos were also cultuted for 24 h, 3 days and 5 days oo glrss coverslips then fixed in formalin and stained with Giemsa. In vitro outgrowth of c&t fPom GCTB fragments Tumour fragments approximately 2 mm in diameter were placed in Fakon tissue adlure flasks, covered with a small amount of culture medium uMEM 10% FCS and incubated at 37 “C in a humidified atmosphere containing 5% COz_ Medium was changed after 24 h and then at 5-7 day intetvak. These cultures were continuously observed and the morphology of cells growing out from the kagntents noted. Cultures were passaged after 6 weeks aod then tegtown and passaged after a futier 2
weeks in culture. At passage 3, cells were frozen in aliquots.
40 In
vivaculture ofGCTB
as subcutaneous
implant
GCTEE
tissue was implanted subcutaneously in athymic MFl nuhu mice. After 40 days, the tumours were excised and fixed in ethanol for embedding in glycol methacrylate. Undecalcified sections were then stained with toluidine blue, van Kossa and for alkaline phosphatase activity and tartrate resistant acid phosphatase activity by using commercial kits (Sigma, UK). A Portion of one of the turnours was fixed in formalin and routinely processed for paraffin embedding. 5 p sections of the above turnours were cut for baematoxylin-eosin, van Kossa and immunoperoxidase staining. Immunohistochemisoy
An indirect immunoperoxidase technique [17] was used to stain cryostat and paraffin-embedded sections of the original turnours, paraffin embedded sections of one of the tumoun which had been implanted subcutaneously into a nude mouse, and glycol metbacrylate embedded sections of another tumour which had been etched in xylene for 15 min. Presence of the proliferating cell nuclear antigen (PCNA) reccgnised by moncclonal antibody PC10 [18], vimentin intermediate filaments, and leucccyte and macrcphage/csteoclsst-associated antigens was determined on the original GCTB outgrowth cells from GCTR fragments, and GCTL3s implanted in nude mice (Table 1). ReSUltS Histology,
hismchemiswy
and immunohismchemisny
oforiginal
GCTBs
All three turnours showed the characteristic histopathological features of a GCTR with prominent mononuclear and giant cell components. Tbe giant ceils showed positive immunobistocbemical staining for CD45, CD68 and vimentin but were negative for HLA-DR and PCNA. CD68 (Fig. la), HLA-DR, CD45 and PCNA (Fig. lb) were expressed by a variable number of mononuclear cells, but almost all cells were positive for vimentin. Histochemical staining of the turncurs showed that the giant cells were positive for tartrate resistant acid pbosphatase and negative for alkaline phcsphatase. The mononuclear cells showed focal alkaline phosphatase positivity particularly amongst spindle cells and only a few scattered cells were tartrate resistant acidphosphatasepositive. Response
to calcitonin
Giant cells freshly isolated from the GCTB specimens all responded to calcitcnin by retraction of cytoplasm and immotility [U]. The degree and duration of this response varied from cell to cell, the duration varying from 5 min to up to 40 min. A minority of mononuclear cells also showed some degree of immotility but it was difficult to determine whether these cells also showed any distinct morphological response to calcitonin. Evidence
ofbone
resorpfion
Resorption pit formation on bone slices was seen in all cultures of cells disaggre-
41 gated from each of the tumours studied. As in previous studies we noted that the longer the period of incubation resorption
pits formed.
on bone slices. the greater the number and size of
Although
some roughening
of the booe auface
but not qulti!wkatcd ah (x412).
was seen
42
43
around smaller (presumed mononuclear) cells, lacunar resorption pits were only seen adjacent to or in the vicinity of large cells greater than 30~ (presumed multinucleated cells).
ofoutgrowth cellosfrom CCTB fragments Adherent cells growing out from the GCTB tissue fragment initially included large multinucleated giant cells and a heterogeneous population of mononuclear cells, some of which were spindle-shaped and fibroblast-like, and others more rounded and macrophage-like with ample cytoplasm (Fig. 2~3). The spindle shaped cells showed occasional mitotic activity and many were positive for HL.A-DR and PCNA and vimentin but negative for LCA and CD& Giant cells persisted for 2 to 3 weeks but were progressively overgrown by the highly proliferative fibroblastic populations (Fig. 2b).
Characteristics
GCTE implants in atkymic mice
days, turnours implanted subcutaneously in athymic mice were evident as small discrete nodules. The tumour was distinct from the subcutaneous tissue of the mouse and had a relatively well-defined but discontinuous fibrous capsule. The tumour implant was composed of the typical cellular components of a GCTB, namely ovoid and spindle-shaped mononuclear cells and scattered multinucleated OSteoclast-like giant cells (Fig. 3a). Many of the mononuclear cellswithm the implants were alkaline phosphatase positive and, in zones of high alkaline phosphatase activity, there was evidence of mineralisation with formation of poorly defined calcific spicules or, in one case, immature bone (both van Kossa positive) (Fig. 3b-d). All giant cells were tartrate-resistant acid phosphatase positive as were scattered mononuclear cells. Immunohiitochemical staining of the tumour implant showed that the giant cells were positive for CD68, CD45 and vlmentin bat negative for I-ILA-DR. Almost allmonottuclearcellswithin theimplatttedtamourstainedforvimetttin and a variable number for CD@ CD45, and HL.A-DR. PCNA was not CXpressed by giant cells in the implant but was strongly expressed by •umc~ol~ rouod and spindle-shaped mononuclear cells. In all cases, the pattern of bnmunohistoChernicaIrextion in the implanted htmottr was similar t0 that Of tba Ot’i!@td htmour. None of the antibodies tested reacted with mouse cells in the epithetiurtt or subcutaneous tissue surrounding the implant. After 40
This study has shown that the mononuclear and multinucleated components ofthe GCTFI are relatively distinct. Multinucleated cells appear to form a ttott-proliferative homogcnecus pop;Zion of cells which show numerous osteoclast-like characteristics. In contrast, the mononuclear cells are composed of a heterogeneous population of cells including cells of macrophage-Iike characteristics, some of which may be mononuclear precursors of the giant cells, and proliferating fibroblast-lie stromal cells, some of which are capable of osteoblastic diiererttiation with strong
44
45
staining for alkaline phosphatase and deposition of woven bone when implanted in viva. Previous studies have shown many similarities between osteoclasts and thP multi-
46 nucleated cells of GCTR. These cells have surface specialisations which resemble a ruffled border [S], are capable of bone resorption [12], respond directly to calcitonin [12], are acid phosphatasc positive [S,7,8] and show a similar pattern of expression of leucocyte (largely macrophage-associated) antigens [9-11,141. In the preseni study, many of these features were also noted in tumour-derived giant cells which were cultured from GCTB fragments and which persisted in subcutaneous implants. In primary cultures, the mdtinucleated cells disappeared after only 2 to 3 weeks, while in the implants, giant cells persisted throughout the 40 day incubation period. Previous cell culture studies have similarly shown that the giant cells, derived by either mechanical/enzymatic dispersion or from outgrowth cultures, do not persist in vitm [24-261 although some investigators have reported formation or persistence of giant cells from mononuclear cells under different culture conditions [27,28]. Byers et al. [27] implanted mononuclear turnour stromal ceUs in a nude mouse but did not observe giant cell formation. These findings, taken together, sug gest that the giant cells are not actively proliferative and cannct be derived, at least in vitro, from the fibroblast-like cells which remain after prolonged culture. Persistence of giant cells in our own implants may indicate that the cellular and tissue architecture of the original turnour must be preserved in order for the giant cells to remain viable. The presence of macrophage-like cells in the manonuclear component of GCTR has previously been established by cytochemical [13], immunohistochemical [9,10,14,1S] and ultrastructural [3-S] studies. These macrophage-like cells, like the giant cells, did not persist in prolonged outgrowth cultures but did remain in implanted turnours where they formed (as in the original turnours) a variable proportion of the mononuclear cell population. Like the giant cells, it would appear that these ceCs form a reactive rather than a neoplastic component of the tumour. This accords with the observations of Burmester et al. [14] and Goldring et al. [24] who showed that cells expressing the macrophage phenotype did not survive in culture, the proliferating cells being devoid of macrophage (including HLA-DR) antigens. The macrophage component within the turnours was immunophenotypically heterogeneous with CD68 (macrophage-associated antigen) and CD45 (Ieucocyte common antigen) positive cells in both the original and implanted turnours showing either expression or absence of HLA-DR. It is possible that mononuclear cells with a similar immunophenotype to giant cells (CD68 positive/HLA-DR negative) may represent the presence of giant celllosteoclast precursors within the original lesion; this is reflected in the transient immotility shown by some of the mononuclear cells in response to calcitonin and the fact that a minority of the mononuclear cells within the original turnour were positive for tartrate resistant acid phnsphatase. The mononuclear cells which persisted in both in vitro and in viva cultures were vimentin positive, CD68 and CD45 negative spindle shaped cells. These cells also formed the proliferative component of the turnour (as assessed by expression of PCNA, a 36-kDa nuclear antigen which is directly involved in DNA synthesis) [la]. In addition, a number of these moronuclanr stmmai cells, both in the original tumow and tumour-derived cell cultures were alkaline phosphatase positive. In implants, evidence of woven bone formation was seen in relation to alkaline phospha-
47 tase positive stromal generally
cells.
show identical
the GClTt
GCTB
to the subcutaneous implants
regarded by some authors as transplants of tumour rather than
in nude mice and are true m&stases
Lung metastases associated with a low-grade
appearances and behaviour
[28]. These features indicate that the proliferative
component
of
is a connective tissue stromal cell which has the capacity to differentiate
along fibroblastic an undifferentiated
and osteoblastic cell lines. This mononuclear mesenchymal
authors who originally
cell of the bone marrow,
stromal ceil may be as proposed by the
described and named this turnour [I], and such a hypothesis
would be in keeping with current ideas on the nature of the stromal cell system in bone [29]. It is not certain whether the stromal cells present in the tumour are responsible for the characteristic giant cell/osteo&st response in GCl’B. But given the close microanatomical and physiological relationship which exists between osteoblasts and osteoclasts, and the fact that a number of giant cell containing lesions of extraskeletal tissues have been reported to contain alkaline phosphatase posjtive mononuclear cells [30-321, it is possible that osteoblastic differentiation of proliferating neoplastic stromal cells results in a cell type which is particularly efficient in recruiting osteoclast precursors from the circulation.
This study was supported in part by grants from the Medical Research Council and the Cancer Research Campaign. We thank Miss L. Watts for typing the manuscript and Mrs M.C. Williamson for expert technical assistance.
3 Steiner CC, Giosh L, D&man HD. Ultrastructure of &ant-~ell hmmm of bate. Hum Pathal 19722:569-w& 4 Hanaoka H, Friedman B, Mack RP. UlrrartrucNre and histogenais of @ant.& tumor of bone. Cancer3970;~1408-1423. 5 Gothlin G, Ericxwn ILE. The asteoclast. Review of ~i~rastruc~~e. ongn, arnl ruucmrs-function re lationship. ClinOnhopRelRea 197~120:201-231. 6 Schajotia F. Giant-cell tumors of bone (osteaclastama), a patholo~isal and histochccl*al study. 1 Bone Joint S.urg 1961;43A:1-29. 7 Ores R. Rosen P. Onh J. Localization of acid phmphataK aniviry ma @ant~ceU tumor of tax. Arch Pathol1%9@54-57. 8 McCarthy FF, Serrano JA, Wasser KNS HL. Dorman HD. The dtrast~~~ral localiiation of secrct~~acidrrhosphatarein~iianr-cell tumorof bone. Clin Onhop 1979;141:2%-x2.
48 12 Chambers TJ. Fuller K, McSbeeby PMJ. The effects of calcium regulating hormones o” bone rewrptie” by isolated human osteoclastomilcells. I Palhol 1985:145:297-305. 13 Wood GW, Neft JR, Gollaho” KA. Gourley WK. Mactophages in giantall
tumours of bone. J W-
,boll977;324:53-58. 14 Bwmester GR, Wincbesrer RI, Dimitriu-Bona
A, Klein M. Steiner G. Sissons HA. Delineation of
four cell types comprising ,he giant eel, tumor of bone. Expression of fa and monocyte-macrophage lineage antisecns.I Cti” Invest 1983:7,:1633-1648. ,5 Kasshara K. Yamamuto T. Ka3ahara A. Giant cell tumor of bone: cytological studies. Br I Cancer 1979;50:201-209. I6 A,ha”asou NA. Wells CA. Quinn 3, Ferguson DP. Heryet A, McGee 30’D. The origin and nature of slromal osteoclast~like mubinucleated giant cells in breast carcinoma: implications for furnow OSleolysis and macrophage biology Br J Cancer 1989;59:491-498. 17 Gat,er KC, Falini B, Mason DY. The use of monoclona, antibodies in histopatholagical diagnosis. I”: Anthony PP, MacSwee” RNM.
eds. Recent Advances in Histopathology No. 12. Edinburgh:
Churchill Livingstone 1984;35-67. 18 Hall PA, Leviso” DA, Woods AL et al. Proliferating cell nuclear anrige” (PCNA)
immunolocalia-
tie” in paraffin seedons: a” index of cell proliferation with evidence of deregulated expression in so)meneoplasmr. I Pathol ,990;162:285-294. 19 Abma”“sbcrger
M. Gsbor” M. Schacer A. Weber K. Antibodies to different intermediate filament
proteins: eell type ,98,:45:427-434.
specific
markers
on
paraffin
embedded
human
tissues.
Lab
fnvest
20 Pulford KAF, Rigney EM, Micklem KJ, Jones M, Stress WP, Getter KC, Mason DY. KP1: a “SW monoclonal antibody that detects a mo”ocy,e,macrophage-arsociared antigen in ,ou,i”ely processed tissue 5ectio”s. 1 Cti” Patho, ! 989;42:414-421. 2, Warnlie RA, Gattcr KC, Falini Bet ill. Diagnosis of human tymphoma with monaclana, anti-leukacyie anfibodies. N Engl I Med l9.S3:309:1275-1281. 22 Naiem M, Gerdes J. Abdulaziz Z. Nash J, Stein H, Mason DY. Production ofmonoclonal antibodies for the immunobistological diagnosis of human lymphoma. I”: Leukaemia Marken al.. eds). New York: Academic Press, 1984;117-125. 23 Chambers TI, 1982:,36:27-39.
Magnus
Cl.
Calsitoni”
allem
behaviour
of
isolated
(Knapp W. et
estec&r,r
J Parho,
24 Goldring SR. Roelke MS, Petriso” KK. Bha” AK. Human giant cell tumors of bone: ide~tificatio” and charaeterisatian of cell types. 1 Clin Invest 1987;79:483-491. 25 Gallardo H. DeLustig ES. Schajowie F. Growth and mainlena”% of hama” gitm, cell hmmrs (osteoclastomas) incontinuousculture. Oncology 1970;24:146. 26 Wienfmub S. Bindenna” I, Somjen D. KLet,erY, Satama R, Weissma” SL. Response to pamthyroid and caleitoni” of human giant cell tumor of bone cubwed in patients’ XN”,. Cl,,, Onhop ,982;,67:227-242. 27 Byers VS. Levi” ASJobnsra” mar antigen-beating
JO. Haeke,, Al. Quantitative immunofluoresce”ee studies of the ,u-
eelI in gia”,
cell tumor of bone and asteogenic sarcoma. Caacer
Res
1Y75:35%20-2528. 28 Mina JM. Bone tu”ws: ~imical, radiological and pathoiogical conela,,o”s. a”dFebigcr. 1989;941-IO?,.
Philadelphia, PA: Lea
29 Owen M. Lineage afastcogenx cells arid their reladonrbip 10 ihe stromal system. In: Peck WA (ed). Bone and Mineral Research “013, Amsrerdam: tlsev!er, ,98>;1--U. 30 Wood GS. Beckstead JH, Turner RR. Hsndricksa” MR. Kempso” RL. Warnke RA. Malignant 6. brow histmcymma tunxx cells resemble fibroblasts. Am I Surg Path 1986;10:323-335. 31 Flanagan AM, Chambers TJ. Osteoclasls are present in the giant cell variant of malignant fibrous ha,,ocytoma.l Pa,holl989:199:53-57. 32 Okajnna K. fmmu”ohi~,ocbemica, and biochemical s,udy of alkai& phosphatase in bane tumeu,a aad,umorousco”ditio”s.
Aeta HislochemCytoehem
198730:41-56.
16(1992)37-48
37
Efsevier BAM
w417
Phenotypic characterisatiou of mononuclear and multinucleated cells of giant cell tumour of bone
C.J. Joyner’, J.M. Quint#, J.T. Triffitt’, M.E. Oven’ and N.A. Athanasou* ‘MRC Bone Researchioboromy. Nuffifd Orthopedic Cmm. Ozfwd and *Nufield Lkpmmm Paholo#y. University of Oxford, John Radcliffe Ho~pml. Oxfonl, UK
of
(Received 18April 1991) (Awepled
lOSeptember 1’9%)
The giant cell tumor- of bone (GCIB) is a relatively uncommon primary bone tumottrwhich usually produces an expansile lytic lesion in the epiphysis and metaphy-
sis of 8 long bone [l?,]. Histo,ogically, these turnours are distinctive, coasisdog of an ovoid or spindle shaped mononuclear stromal cell component amongst which are scattered osteoclast-like multinucleated giant cells. There has been considerable
38
controversy surrounding the nature of the two cellular components. In particular, the identity of the cell type(s) forming the mononuclear cell component, the relationshia of the mononuclear stromal cells to the osteoclast-like giant cells and the nature of the proliferating cell in GCfB have not been clarified. The giant cells have numerous ultrastructural [3-S], cytochcmical [S-B], immunohistochemical [911] and functional [i2] features in common with the osteoclast. The mononuclear cell component is heterogeneous and composed of macrophages, fibroblast-like stromal cells and cells intermediate between these two types [3,4,13-151. In this study, we have sought to define further the nature of the cell types present in both the mononuclear and giant cell components of GCI’B at the time of resection and following culture in vitro and as subcutaneous implant in athymic mice. Cells from 3 cases of human GClB have been analysed in terms of their morphology, antigenic phenotype, cytochemistry and functional characteristics. These features have been compared with those of macrophages, osteoclasts and osteoblasts in order to establish whether there is a possibkcrelationship between these cells and the mor.o- or multinucleated cells of the GCTB.
Materials and Methods
Three GCTBs were examined in this study. Two were removed from the proximal femur, and one from the proximal humerus of patients aged 21, SOand 22 years respectively. Samples of the tumour were snap-frozen and stared in liquid nitrogen for cryostat sectioning; samples were also fued in formalin for routine histological processing and staining to confirm the diagnosis of GCTB. The rest of the tumour was either used fresh for functional studies, in vitro culture and in vivo culture as subcutaneous implant or frozen down for subsequentUSCin each of these types of study. Tissue for freezing was first cut int;, tiny pieces (2 nuns) then incubated in FCS (Gibw) containing 10% diiethylsulphoxide (DMSO) {IS min; UT) and finally cooled to -WC using a programmable ce.Ufreezer (Planer, Middlesex, UK). Samples were stored at -13YC until required for culture. On removal, the tumour was rinsed repeatedly in serum-free medium to remove the DMSO. of bone resorption Fresh pieces of tumour tissue were placed in RPM1 1640 tissue culture medium (Gibco) supplemented with antibiotics and 10% fetal calf strum (RPMUFCS). Portions of the tumour were then placed in a 35 mm Petri dish (Sterilin) and finely curetted with a scalpel blade.. The tissue suspension was then agitated with a glass pipette, heavier pieces allowed to settle and the suspension then pipetted into 16 mm wells of a Costar multiwell plate which contained 15 mm glass coverslips or cortical bone slices prepared as previously described [14. The cell suspension was settled on bone slices or coverslips for 30 min and then washed in RMFW’PCSand placed in fresh wells containing tissue culture medium. Cells cultured on bone slices and coverslips were incubated for up to 5 days, being removed after 24 h, 3 days and 5 days,
Response to calcitotdn and evidence
39 Table 1 Mottoclonal antibodies used in this study and immunoreactivity of ttmtott~lear cells (MO) and multinucleated giant cells (GCs) in original turnours and subctttaneous implants
PC10
I1
v9
1191
KPI PD7a6
IN
CR3143
Proliferaling cell nuclear antigen vimentin infcmlcdias fiaments CD68
+
WI
CD45(kVc-xy?C unnmonantipn)
1221
HLADR’
+
++
++
+
++
+
i+
at which time one of a pair of bone slices was fixed in 5% glutaraklehyde; the other was treated with Ttiton X-100 (0.1% in distilled water) to remove the cells from the bone surface and enable the full extent of resorption pit formation to be observed by scanning electron microcopy. Bone slices were dehydrated through a graded ethanol series, critical point dried from CO*, sputter coated with gold then examined in a Philips SEM 505 scatuting electron microscope. One of a pair of glass coverslips containing cell suspension was placed immediately after washing in tissue culture medium containing 1 &ml salmon calcitonin (APbammcmticals, Eastboume). The cakitonbt response ofmultinucleatcd and moaonuckar cells was observed continuously for up to 1 h and the response compared with cells on coverslips incubated in tissue culture medium alone. Cell suspensions ozt covetslips were fixed bt fotmalii after 1 h and stained with Giemsa. Cell suspensioos were also cultuted for 24 h, 3 days and 5 days oo glrss coverslips then fixed in formalin and stained with Giemsa. In vitro outgrowth of c&t fPom GCTB fragments Tumour fragments approximately 2 mm in diameter were placed in Fakon tissue adlure flasks, covered with a small amount of culture medium uMEM 10% FCS and incubated at 37 “C in a humidified atmosphere containing 5% COz_ Medium was changed after 24 h and then at 5-7 day intetvak. These cultures were continuously observed and the morphology of cells growing out from the kagntents noted. Cultures were passaged after 6 weeks aod then tegtown and passaged after a futier 2
weeks in culture. At passage 3, cells were frozen in aliquots.
40 In
vivaculture ofGCTB
as subcutaneous
implant
GCTEE
tissue was implanted subcutaneously in athymic MFl nuhu mice. After 40 days, the tumours were excised and fixed in ethanol for embedding in glycol methacrylate. Undecalcified sections were then stained with toluidine blue, van Kossa and for alkaline phosphatase activity and tartrate resistant acid phosphatase activity by using commercial kits (Sigma, UK). A Portion of one of the turnours was fixed in formalin and routinely processed for paraffin embedding. 5 p sections of the above turnours were cut for baematoxylin-eosin, van Kossa and immunoperoxidase staining. Immunohistochemisoy
An indirect immunoperoxidase technique [17] was used to stain cryostat and paraffin-embedded sections of the original turnours, paraffin embedded sections of one of the tumoun which had been implanted subcutaneously into a nude mouse, and glycol metbacrylate embedded sections of another tumour which had been etched in xylene for 15 min. Presence of the proliferating cell nuclear antigen (PCNA) reccgnised by moncclonal antibody PC10 [18], vimentin intermediate filaments, and leucccyte and macrcphage/csteoclsst-associated antigens was determined on the original GCTB outgrowth cells from GCTR fragments, and GCTL3s implanted in nude mice (Table 1). ReSUltS Histology,
hismchemiswy
and immunohismchemisny
oforiginal
GCTBs
All three turnours showed the characteristic histopathological features of a GCTR with prominent mononuclear and giant cell components. Tbe giant ceils showed positive immunobistocbemical staining for CD45, CD68 and vimentin but were negative for HLA-DR and PCNA. CD68 (Fig. la), HLA-DR, CD45 and PCNA (Fig. lb) were expressed by a variable number of mononuclear cells, but almost all cells were positive for vimentin. Histochemical staining of the turncurs showed that the giant cells were positive for tartrate resistant acid pbosphatase and negative for alkaline phcsphatase. The mononuclear cells showed focal alkaline phosphatase positivity particularly amongst spindle cells and only a few scattered cells were tartrate resistant acidphosphatasepositive. Response
to calcitonin
Giant cells freshly isolated from the GCTB specimens all responded to calcitcnin by retraction of cytoplasm and immotility [U]. The degree and duration of this response varied from cell to cell, the duration varying from 5 min to up to 40 min. A minority of mononuclear cells also showed some degree of immotility but it was difficult to determine whether these cells also showed any distinct morphological response to calcitonin. Evidence
ofbone
resorpfion
Resorption pit formation on bone slices was seen in all cultures of cells disaggre-
41 gated from each of the tumours studied. As in previous studies we noted that the longer the period of incubation resorption
pits formed.
on bone slices. the greater the number and size of
Although
some roughening
of the booe auface
but not qulti!wkatcd ah (x412).
was seen
42
43
around smaller (presumed mononuclear) cells, lacunar resorption pits were only seen adjacent to or in the vicinity of large cells greater than 30~ (presumed multinucleated cells).
ofoutgrowth cellosfrom CCTB fragments Adherent cells growing out from the GCTB tissue fragment initially included large multinucleated giant cells and a heterogeneous population of mononuclear cells, some of which were spindle-shaped and fibroblast-like, and others more rounded and macrophage-like with ample cytoplasm (Fig. 2~3). The spindle shaped cells showed occasional mitotic activity and many were positive for HL.A-DR and PCNA and vimentin but negative for LCA and CD& Giant cells persisted for 2 to 3 weeks but were progressively overgrown by the highly proliferative fibroblastic populations (Fig. 2b).
Characteristics
GCTE implants in atkymic mice
days, turnours implanted subcutaneously in athymic mice were evident as small discrete nodules. The tumour was distinct from the subcutaneous tissue of the mouse and had a relatively well-defined but discontinuous fibrous capsule. The tumour implant was composed of the typical cellular components of a GCTB, namely ovoid and spindle-shaped mononuclear cells and scattered multinucleated OSteoclast-like giant cells (Fig. 3a). Many of the mononuclear cellswithm the implants were alkaline phosphatase positive and, in zones of high alkaline phosphatase activity, there was evidence of mineralisation with formation of poorly defined calcific spicules or, in one case, immature bone (both van Kossa positive) (Fig. 3b-d). All giant cells were tartrate-resistant acid phosphatase positive as were scattered mononuclear cells. Immunohiitochemical staining of the tumour implant showed that the giant cells were positive for CD68, CD45 and vlmentin bat negative for I-ILA-DR. Almost allmonottuclearcellswithin theimplatttedtamourstainedforvimetttin and a variable number for CD@ CD45, and HL.A-DR. PCNA was not CXpressed by giant cells in the implant but was strongly expressed by •umc~ol~ rouod and spindle-shaped mononuclear cells. In all cases, the pattern of bnmunohistoChernicaIrextion in the implanted htmottr was similar t0 that Of tba Ot’i!@td htmour. None of the antibodies tested reacted with mouse cells in the epithetiurtt or subcutaneous tissue surrounding the implant. After 40
This study has shown that the mononuclear and multinucleated components ofthe GCTFI are relatively distinct. Multinucleated cells appear to form a ttott-proliferative homogcnecus pop;Zion of cells which show numerous osteoclast-like characteristics. In contrast, the mononuclear cells are composed of a heterogeneous population of cells including cells of macrophage-Iike characteristics, some of which may be mononuclear precursors of the giant cells, and proliferating fibroblast-lie stromal cells, some of which are capable of osteoblastic diiererttiation with strong
44
45
staining for alkaline phosphatase and deposition of woven bone when implanted in viva. Previous studies have shown many similarities between osteoclasts and thP multi-
46 nucleated cells of GCTR. These cells have surface specialisations which resemble a ruffled border [S], are capable of bone resorption [12], respond directly to calcitonin [12], are acid phosphatasc positive [S,7,8] and show a similar pattern of expression of leucocyte (largely macrophage-associated) antigens [9-11,141. In the preseni study, many of these features were also noted in tumour-derived giant cells which were cultured from GCTB fragments and which persisted in subcutaneous implants. In primary cultures, the mdtinucleated cells disappeared after only 2 to 3 weeks, while in the implants, giant cells persisted throughout the 40 day incubation period. Previous cell culture studies have similarly shown that the giant cells, derived by either mechanical/enzymatic dispersion or from outgrowth cultures, do not persist in vitm [24-261 although some investigators have reported formation or persistence of giant cells from mononuclear cells under different culture conditions [27,28]. Byers et al. [27] implanted mononuclear turnour stromal ceUs in a nude mouse but did not observe giant cell formation. These findings, taken together, sug gest that the giant cells are not actively proliferative and cannct be derived, at least in vitro, from the fibroblast-like cells which remain after prolonged culture. Persistence of giant cells in our own implants may indicate that the cellular and tissue architecture of the original turnour must be preserved in order for the giant cells to remain viable. The presence of macrophage-like cells in the manonuclear component of GCTR has previously been established by cytochemical [13], immunohistochemical [9,10,14,1S] and ultrastructural [3-S] studies. These macrophage-like cells, like the giant cells, did not persist in prolonged outgrowth cultures but did remain in implanted turnours where they formed (as in the original turnours) a variable proportion of the mononuclear cell population. Like the giant cells, it would appear that these ceCs form a reactive rather than a neoplastic component of the tumour. This accords with the observations of Burmester et al. [14] and Goldring et al. [24] who showed that cells expressing the macrophage phenotype did not survive in culture, the proliferating cells being devoid of macrophage (including HLA-DR) antigens. The macrophage component within the turnours was immunophenotypically heterogeneous with CD68 (macrophage-associated antigen) and CD45 (Ieucocyte common antigen) positive cells in both the original and implanted turnours showing either expression or absence of HLA-DR. It is possible that mononuclear cells with a similar immunophenotype to giant cells (CD68 positive/HLA-DR negative) may represent the presence of giant celllosteoclast precursors within the original lesion; this is reflected in the transient immotility shown by some of the mononuclear cells in response to calcitonin and the fact that a minority of the mononuclear cells within the original turnour were positive for tartrate resistant acid phnsphatase. The mononuclear cells which persisted in both in vitro and in viva cultures were vimentin positive, CD68 and CD45 negative spindle shaped cells. These cells also formed the proliferative component of the turnour (as assessed by expression of PCNA, a 36-kDa nuclear antigen which is directly involved in DNA synthesis) [la]. In addition, a number of these moronuclanr stmmai cells, both in the original tumow and tumour-derived cell cultures were alkaline phosphatase positive. In implants, evidence of woven bone formation was seen in relation to alkaline phospha-
47 tase positive stromal generally
cells.
show identical
the GClTt
GCTB
to the subcutaneous implants
regarded by some authors as transplants of tumour rather than
in nude mice and are true m&stases
Lung metastases associated with a low-grade
appearances and behaviour
[28]. These features indicate that the proliferative
component
of
is a connective tissue stromal cell which has the capacity to differentiate
along fibroblastic an undifferentiated
and osteoblastic cell lines. This mononuclear mesenchymal
authors who originally
cell of the bone marrow,
stromal ceil may be as proposed by the
described and named this turnour [I], and such a hypothesis
would be in keeping with current ideas on the nature of the stromal cell system in bone [29]. It is not certain whether the stromal cells present in the tumour are responsible for the characteristic giant cell/osteo&st response in GCl’B. But given the close microanatomical and physiological relationship which exists between osteoblasts and osteoclasts, and the fact that a number of giant cell containing lesions of extraskeletal tissues have been reported to contain alkaline phosphatase posjtive mononuclear cells [30-321, it is possible that osteoblastic differentiation of proliferating neoplastic stromal cells results in a cell type which is particularly efficient in recruiting osteoclast precursors from the circulation.
This study was supported in part by grants from the Medical Research Council and the Cancer Research Campaign. We thank Miss L. Watts for typing the manuscript and Mrs M.C. Williamson for expert technical assistance.
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