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Pathological significance of cilia and centrioles in bone and soft tissue tumors
J Orthop Sci (1997) 2:137-145
~
lournalof
thopaedic Science TheJapanese Orthopaedic Association
Pathological significance of cilia and centrioles in bone and soft tissue, tumors GERSON GANDHI GANEV Teikyo University School of Medicine, Department of Orthopaedic Surgery, 2-11-I Kaga, Itabashi-ku, Tokyo 173, Japan
Abstract: The incidence and ultrastructure of rudimentary cilia and free centrioles were examined with a transmission electron microscope in 9481 cells from 74 bone and soft tissue tumors (benign and malignant) and from 11 samples of normal, histologically related tissues. The cilia were rudimentary (nonmotile), with a 9 + 0 axonemal pattern (occasionally 7 + 0 or 5 + 0), lacking the central tubules and two dynein arms of subfiber A, and with elongated shafts and basal structures consisting of two centrioles. Free centrioles appeared in all tissues studied, in general close to the Golgi apparatus. There was a significantly higher incidence of cilia and centrioles in the malignant tumors, compared to the incidence in benign tumors and normal tissues, and there was an inverse correlation between the incidence of cilia and centrioles in malignant tumors. Centrioles were not observed in ciliated cells, nor were cilia observed in cells undergoing mitosis. These findings suggest that neoplasia can lead to an increase in the incidence of cilia and centrioles, and that the increase correlates with the malignancy of a tumor. Consideration of the incidence of cilia and centrioles probably gives a better idea of cell proliferation than consideration of the incidence of cilia only. Key words: bone and soft tissue tumors, ultrastructure, cilia
Introduction The presence of cilia has been reported in a variety of tissues throughout the animal kingdom. Cilia in animal tissues can be motile or immotile. The latter are also
Offprint requests to: G.G. G a n e v Received for publication on Aug. 2, 1996; accepted on Dec. 10, 1996
called oligocilia, primary cilia, rudimentary cilia, or solitary cilia. Motile and immotile cilia differ in their m o d e of morphogenesis, ultrastructure, and function. Motile cilia have a "9 + 2" axonemal pattern, which consists of nine doublet microtubules in the peripheral area and two singlet microtubules in the central area. The doublet microtubules are c o m p o s e d of subfiber A, with two dynein arms involving ATPase, and subfiber B. Motile cilia can undergo pathological ultrastructural changes in a variety of diseases, 1,4,1~ though some changes may be reversible. 1,z2 C o m p a r e d with motile cilia, rudimentary cilia were r e p o r t e d to lack the central pair of microtubules, resulting in a "9 + 0" structure. Further, Park et al. 2~reported that two dynein arms of subfiber A were absent in r u d i m e n t a r y cilia of Schwann cells of a malignant schwannoma, and that rudimentary cilia were immotile because of the absence of the dynein arms. Most vertebrate cells produce apparently nonfunctional, rudimentary cilia, which have b e e n observed in a variety of tissues, such as smooth muscle, brain, liver, lungs, spleen, and testis? R u d i m e n t a r y cilia also occur in carcinomas 5 and sarc o m a s ? Park et al? ~ reported a higher incidence of cilia in malignant peripheral nerve tumors than in benign tumors. They concluded that this feature could possibly be an ultrastructural m a r k e r for cell proliferation in tumors. They also noted that cilia a p p e a r e d to be closely associated with cell division in nerve tumors, and that the incidence of cilia was inversely correlated with that of free centrioles. However, the incidence and relationship of cilia and free centrioles in other types of tumors have not yet been studied. In this investigation, we investigated the incidence of cilia and free centrioles in bone, cartilage, and nerve tumors, as well as in normal tissues histologically related to the tumors. The purpose of this study was to establish the pathological significance of cilia and centrioles in t u m o r cells.
138
G.G. Ganev: Cilia and centrioles in tumors
Materials and m e t h o d s F r e s h tissues f r o m 8 o s t e o s a r c o m a s , 6 E w i n g ' s s a r c o m a s of bone, 9 chondrosarcomas, 9 malignant schwannomas, 14 e n c h o n d r o m a s , 20 b e n i g n p e r i p h e r a l n e r v e t u m o r s (neurofibroma and schwannoma), and 8 miscellaneous t u m o r s w e r e o b t a i n e d at surgery. N o r m a l tissues (3 s a m p l e s o f histologically n o r m a l sciatic n e r v e , 1 o f nor-
m a l n e r v e in skin tissue, an d 8 o f n o r m a l c a r t i l a g e ) w e r e o b t a i n e d f r o m a m p u t a t e d limbs. A l l tissues w e r e cut into small p i e c e s with a r a z o r blade. T h e m a t e r i a l s w e r e t h e n i m m e r s e d in 2.5% g l u t a r a l d e h y d e and 2 % p a r a f o r m a l d e h y d e , b u f f e r e d with 0 . 1 M c a c o d y l a t e b u f f e r at p H 7.4, an d w e r e k e p t o v e r n i g h t at 4~ A f t e r wards, t h e y w e r e r i n s e d s e v e r a l t i m es with t he s a m e buffer, an d t h e n e x p o s e d to 1% b u f f e r e d o s m i u m
Table 1. Tumors and related tissues examined No. of cases examined
Cell type
No. of cells examined
Osteosarcoma Osteosarcoma Osteosarcoma Ewing's sarcoma of bone Chondrosarcoma Enchondroma Normal cartilage (growth plate) Malignant schwannoma Schwannoma Neurofibroma Normal nerve Primitive neuroectodermal tumor Liposarcoma Intramuscular myxoma Malignant hemangiopericytoma Clear cell sarcoma Chordoid sarcoma Epithelioid sarcoma
2 5 1 6 9 14 8 9 8 12 3 1 2 1 1 1 1 1
Osteoblast Chondroblast Fibroblast Tumor cell Chondroblast Chondroblast Chondrocyte Schwann cell Schwann cell Schwann cell Schwann cell Tumor cell Lipoblastic cell Tumor cell Endothelial cell Tumor cell Tumor cell Tumor cell
114 449 99 452 1007 1445 605 2107 970 868 557 77 180 67 200 100 98 86
Total
85
Tissues
9481
Table 2. Incidence a of cilia and centrioles and statistical evaluation in tumors and related tissues Tissues
No. of cases
Osteosarcoma Ewing's sarcoma of bone Chondrosarcoma Enchondroma Normal chondrocytes from cartilage (growth plate) Malignant schwannoma Benign nerve tumors Normal nerve Miscellaneous tumors
1:4 1:7 1:16 4:7 4:16 7:16 7:10 7:13 10:13 16:19
NS P < 0.01 NS P < 0.01 NS NS P<0.01 P<0.01 NS P<0.01
Percentage of cells with cilia
8 6 9 14 8
1. 4. 7. 10. 13.
9 20 3 8
16. 5.9 _+ 4.2 19. 1.1 _+ 1.2 0 5.8
2:5 P < 0.05 3:6 NS 2:8 P < 0.01 3:9 P < 0.05 2:17 P < 0 . 0 5 3:18 NS 5:8 NS 6:9 P < 0.01 5:17 NS 6:18 P<0.05 8:17 NS 9:18 NS 8:11 P<0.01 9:12 P<0.01 8:14 NS 9:15 P<0.01 11:14 NS 12:15 NS 17:20 P<0.01 18:21 P<0.01 17:22 P < 0 . 0 5 18:23 P<0.05 20:22 NS 21:23 P < 0.05 NS, Non-significant. Comparison was done with the Wilcoxon test. aMean values _+ SD (for miscellaneous tumors, mean value only)
2.5_+ 1.2 3.0_+2.0 8.1 _+ 3.3 2.0 _+ 1.6 2.4 _+ 2.5
Percentage of cells with centrioles 2. 5. 8. 11. 14.
4:10 7:19 10:16 8:19
Percentage of cells with cilia and with centrioles
7 . 2 + 1.9 4.8_+2.3 4.1 _+ 3.6 1.6 _+ 0.9 2.4 _+ 1.9
3. 9.7 + 2 . 0 6. 7.7_+2.2 9. 12.2 _+ 2.2 12. 3.6 _+ 1.7 15. 4.8 _+ 2.6
17. 4.6 _+ 2.6 20. 1.6 _+ 1.4 22. 0.1 _+ 0.2 4.6
18. 10.5 + 1.9 21. 2.6 _+ 1.8 23. 0.1 +_ 0.2 10.4
NS P<0.01 P<0.01 NS
G.G. Ganev: Cilia and centrioles in tumors tetroxide for 2h. Some specimens were fixed by the tetra-fixation method, using glutaraldehyde, tannic acid, and osmium tetroxide. 21Some tissues from bone tumors were fixed with a mixture of 1% glutaraldehyde and 4% paraformaldehyde in 0.1M cacodylate buffer for 2448h at room temperature. After prefixation, the bone tumors were treated with 5% ethylene diamine tetraacetic acid ( E D T A ) for 1-2 weeks at room temperature, and then postfixed with osmium tetroxide. Some cartilaginous tissues (such as enchondroma and normal cartilage) and some l~erve tumors were fixed with a mixture of osmium tetroxide and potassium ferrocyanide, according to the method described by Schnepf et al., 24 after aldehyde fixation. All specimens were dehydrated with ethanol and embedded in Epon 812, Quetol 812, and Spurr resin. Ultrathin sections cut from the resin blocks were stained with uranyl acetate and lead citrate. The stained sections were observed with J E O L (Tokyo, Japan) 100 S and 1200EX electron microscopes. Electron microscopic negatives were taken at random at x2000 magnification from multiple areas of many sections from approximately 800 blocks. The negatives were enlarged at x 1 0 0 0 0 - x 1 8 0 0 0 and printed on large photographic paper, and every cell in the prints was studied individually, using a loupe to identify cilia and centrioles. Before determining the incidence of cilia we examined the cell type in each sample, and only cells that characterized the tumor (for example, osteoblasts in a osteoblastic type osteosarcoma) were entered in this:study. The cells and tissues studied are summarized in Table 1. For statistical analysis, the mean percentage and standard deviation were calculated for the incidence of cilia and centrioles in each group of benign tumors, malignant tumors, and normal cells. The values were then estimated with the Wilcoxon test; P < 0.05 was considered a significant difference. Overall statistical results are summarized in Table 2. Benign nerve tumors (schwannoma and neurofibroma) were statistically analyzed as one group. The incidence of cilia and centrioles in the miscellaneous tumors was examined, but not included in the general statistical analysis.
139 quently, in longitudinal profile (Figs. 6, 7). Most cilia were seen at the cell periphery, but some were situated deep in the cytoplasm in close association with invaginated cell membranes (Fig. 8). The ultrastructure of rudimentary cilia was similar in benign and malignant tumors, and also in normal cells, such as normal chondrocytes. The cilia consisted of long shafts with a 9 + 0 axonemal pattern (Fig. 2), but other patterns also appeared, such as 8 + 0, 7 + 0, 6 + 0, and 5 + 0 (Fig. 3). Each of the nine peripheral doublets consisted of subfibers A and B. The two dynein arms of subfiber A, normally present in motile cilia, were absent in the cilia of osteosarcoma, chondrosarcoma, malignant schwanoma, clear cell sarcoma, and normal chondrocytes (Fig. 2). At the base of the cilium, a basal body, consisting of two centrioles, was present, attached to the end of the axonemes (Figs. 7, 9). The transitional fibers emerging from the basal body were connected with cell mem-
Fig. 1. In malignant schwannoma, four rudimentary cilia were found in the extracellular space in the vicinity of a Schwann cell membrane
Results
Ultrastructural findings Rudimentary cilia usually occurred singly in tumor cells and the related normal cells, except for Schwann cells of normal nerve, where cilia were not found. In some Schwann cells of malignant schwannoma, several cilia per cell were seen (Fig. 1). Cilia were sometimes seen in transverse and oblique profiles (Figs. 1-5) or, less fre-
Fig. 2. Transverse profile of a rudimentary cilium in an osteoblast of osteosarcoma. The cilium has a 9 + 0 axonemal pattern of microtubules. Note that two dynein arms are absent in subfiber A of the cilium (arrowhead)
140
Fig. 3. Rudimentary cilium with a 5 + 0 axonemal pattern of
G.G. Ganev: Cilia and centrioles in tumors branes. The basal body contained well developed adnexa, such as triangular basal feet and some long striated rootlets (Figs. 8, 9). Two basal feet could be seen in the side walls of a basal body (Figs. 7, 9). A free centriole was usually observed in tumor cells and in cells of normal tissues, in general near the Golgi apparatus (Fig. 10). Cilia and free centrioles were not observed in the same cell. When cilia were found in the cells examined, these ciliated cells contained no centrioles, and when centrioles were found in the cells, no cilia were present. Moreover, no cilia were observed in cells undergoing mitosis, although centrioles usually appeared in the mitotic cells.
a Schwann cell in malignant schwannoma
Fig. 4. Ciliary shaft (arrowhead) in an osteoblast of osteosarcoma
Fig. 5. Rudimentary cilium chondroblast in enchondroma
of
a
G.G. Ganev: Cilia and centrioles in tumors
141
Statistical analysis Incidence of cilia. Chondrosarcoma and malignant
Fig. 6. Ciliated chondroblast in chondrosarcoma
schwannoma were the tumor groups showing the highest incidence of cilia, and osteosarcoma and Ewing's sarcoma showed the next highest incidence (Table 2; Fig. 11). The difference between chondrosarcoma and osteosarcoma in incidence of cilia was significant and the difference between chondrosarcoma and Ewing's sarcoma was also significant, although there was no significant difference between malignant schwannoma and osteosarcoma, or between malignant schwannoma and Ewing's sarcoma (Table 2). Compared to the benign tumors, the malignant tumors usually had a higher incidence of cilia (Table 2; Fig. 11); there were significant differences between
Fig. 7. Typical rudimentary cilium in clear cell sarcoma. The cilium has basal bodies, a long elongated ciliary shaft, and triangular basal feet structures
Fig. 8. Ciliary shaft (oblique profle) seen in a Schwann cell in malignant schwannoma. The cytoplasm in the vicinity of the ciliary base involves a rootlet structure (arrowheads)
Fig. 9. Basal bodies in a Schwann cell of a neurofibroma, showing two triangular basal feet structures (arrowheads)
142
G.G. Ganev: Cilia and centrioles in tumors
Fig. 10. Free centriole in an osteoblast of osteosarcoma
9 8 7 6 5 4 3 2 1 0 NN
NC
EC
SW
NF
MS
CS
OS
EW
Fig. 11. Average incidence of cilia in the tissues studied. NN, Normal nerve; NC, normal chondrocyte; EC, enchondroma; SW, Schwannoma; NF, neurofibroma; MS, malignant schwannoma; CS, chondrosarcoma; OS, osteosarcoma; EW, Ewing's sarcoma
malignant schwannoma and benign nerve tumors, between chondrosarcoma and enchondroma, between chondrosarcoma and benign nerve tumors, and between malignant schwannoma and enchondroma. The benign nerve tumors had a higher incidence of cilia than the normal nerves, whereas enchondromas and normal nerves showed no difference in incidence. Normal chondrocytes showed a higher incidence of cilia than enchondromas (Table 2); this might have been because the cartilage samples examined were from the growth plate, where cell proliferation is higher than in normal cartilage. In the normal tissues examined, normal chondrocytes often had cilia, as did the tumor cells of enchondroma, but Schwann cells of normal nerve did not have any cilia.
Incidence of free centrioles. Among malignant tumors, osteosarcoma and Ewing's sarcoma had the highest incidence of free centrioles, followed by malignant schwannoma and chondrosarcoma; osteosarcoma showed an especially high incidence, and the difference between this tumor and all other types of malignant tumors was significant (Table 2, Fig. 12). Differences among Ewing's sarcoma, chondrosarcoma, and malignant schwanoma were not significant. Enchondroma and benign nerve tumors showed a low incidence of centrioles. Normal cartilage showed a higher incidence than benign tumors, but the cartilage tissue studied here was from areas involving the growth plate, which differs from articular cartilage. Normal nerve had the lowest incidence of centrioles. Overall, malignant tumors consistently showed a higher incidence of centrioles than benign tumors and normal tissues (Table 2, Fig. 12): the difference was significant for both benign tumors and normal tissues. However, the difference in incidence of free centrioles between benign tumors and normal tissues was not significant (Table 2). Combined incidence of cells with cilia and cells with free centrioles. The combined incidence of ciliated cells and cells with free centrioles showed three apparently distinct tissue groups: malignant tumors, which had a high incidence (probably due to the highest proliferative activity), benign tumors and the cartilage growth plate, which had a moderate incidence, and normal tissue, which had a low incidence (Table 2, Fig. 13). Functionally, the centriole is the "common component" shared by cilia and free centrioles (since the ciliary basal body is formed by the centrioles), so the combined incidence of both structures corresponds to the incidence of
G.G. Ganev: Cilia and centrioles in tumors
143
centrioles that arise from the basal bodies of cilia and those that arise from the free centrioles of the cytoplasm. Miscellaneous tumors, which consisted mostly of sarcomas, showed a high incidence of both cilia and centrioles (Table 2). In malignant tumors, we found a negative correlation coefficient between the incidence of cilia and centrioles; this indicates that a high incidence of cilia in a malignant tumor was associated with a low incidence of centrioles, and a high incidence of centrioles was associated with a low incidence of cilia. This was not observed in benign tumors or in normal cells.
Ciliated cells have been documented in many neoplasms, including peripheral nerve tumors; ~9,2~gliomas; 26 ependymomas; 23 adenocarcinomas of the stomach, uterus, and pancreas; 5,7,~8 endometrial carcinoma; 9 and mesothelioma? 7 Further, ciliary abnormalities have been described in inflammatory conditions, including chronic respiratory diseases 4~22,28 and glomerulonephritis. 1~ The opportunity to encounter cilia and free centrioles in thin sections is rare, and analysis of the incidence depends on observations confined to small areas of specimens. Therefore, in this study, we attempted to observe more samples from each subject, containing as many ceils as possible. Additionally, we examined some tissues in which the presence of cilia, to the best of our knowledge, has not been documented before, such as bone sarcomas and enchondroma. Cilia found in tumors are mostly of the rudimentary (immotile) type, with a 9 + 0 axonemal pattern? ,~9,2~In this study, we found variations of the traditional pattern of axonemes (9 + 0) in rudimentary cilia, including 8 + 0, 7 + 0, 6 + 0, and 5 + 0 patterns; this may indicate that the cilia were cut at different heights in the ciliary shaft?
We also found cilia in all tissues studied, except in Schwann cells of normal nerve. We believe that cilia are distributed in all tissues or cells, including normal, inflammatory, and neoplastic. The occurrence of rudimentary cilia has been explained in different ways. Torikata et al. 27 reported ciliated cells in a malignant pleural mesothelioma and suggested that mesothelial cells could be changed into ciliated cells in the process of tumorigenesis. Bray 2 suggested that the cell "program" for making a cilium could be activated in many different situations in which an extension of the cell surface was needed, even if movement was not required. Park et al., 2~ describing an increased incidence of cilia in peripheral nerve tumors, emphasised the inverse correlation betv)een cilia formation and mitosis, and the absence of cilia in mitotic cells, despite the presence of cilia in highly proliferative tissues. Milhaud and Papas 17 induced ciliogenesis in cat brain cells with pargyline (a m o n o a m i n e oxidase inhibitor) and suggested that centriolar production without subsequent mitosis may lead to the production of cilia. The relationship between cilia and centrioles, and between centrioles and cell division, is the key to clarifying the role of cilia and centrioles in tumor cells: cilia arise from basal bodies, and basal bodies from centrioles. 2 Centrioles are part of the cell centrosome, which is the major microtubule-organizing center in most animal cells, involved in cell m o v e m e n t and cell division. 2 Rudimentary cilia are formed by one of the interphase centrioles, and the occasional presence of aberrant intracytoplasmic cilia (common in ependymomas) has been interpreted as a failure in the migration of the basal body to the p r o p e r apical surface of the cell. 12,15Centriole ciliation has been shown to precede D N A synthesis by 5--6h; 13 accordingly, Katsumoto et al. TM have suggested that the organization of cilia plays an important role in controlling the cell's commitment to initiation of D N A synthesis.
JCentrioles% J
I Cilia+Centrioles%
Discussion
I
20
8765-
10
432
10-
0 NN
NC
EC
SW
NF
MS
CS
06
EW
Fig. 12. Average incidence of free centrioles in the tissues studied. For abbreviations, see Fig. 11
NN
NC
~
EC
SW
NF
MS
CS
OS
EW
Fig. 13. Average incidence of ciliate cells plus cells with free centrioles. For abbreviations, see Fig. 11
144 We found a higher incidence of cilia in malignant than in benign tumors; and this can be interpreted as a sign of high cell proliferation in malignant tumors; the association of cilia with poor tumor differentiation was not clear in this study, although it has been reported before. 19Cilia and centrioles were not found in the same cell in our study, and we found a negative correlation between the incidence of cilia and centrioles in malignant tumors. Cilia and free centrioles appeared at different stages of the cell cycle (centrioles being "consumed" in the formation of the ciliary basal body), and the relationship between cilia and centrioles was more clearly expressed in malignant tumors, where cell proliferation was probably higher. Osteosarcoma is the most c o m m o n malignant bone tumor, followed by chondrosarcoma and Ewing's sarcoma? The incidence of cilia was not high in osteosarcoma and Ewing's sarcoma of bone. On the other hand, these tumors had the highest incidence of centrioles of all tissues studied. This may indicate a high mitotic rate in these tumors, which could explain both findings i.e., the low incidence of cilia and the high incidence of centrioles (cilia are not found in mitotic cells). The difference in the cilia-centriole profile between osteosarcoma and chondrosarcoma (which had the highest incidence of cilia among all tissues studied) may help to explain why sensibility to chemotherapy is different in these tumors, with osteosarcoma probably having a larger growth fraction (percentage of proliferating cells in a tumor). Analysis of the incidence of both cilia and centrioles may provide a more precise indication of tumor proliferation, since both structures seem to be involved in cell division at different stages of the cell cycle. We found that normal tissue with high proliferation (cartilage growth plate) had a high incidence of centrioles, even higher than that seen in benign tumors (Table 2, Fig. 12). Cell kinetics would seem to differ greatly between benign (Schwannoma, neurofibroma) and malignant nerve tumors (malignant schwannoma), since differences in the incidence of both cilia and centrioles were highly significant ( P < 0.01). Differences between benign nerve tumors and normal nerve were not as significant, and it seems that benign tumors retain most of the normal, low proliferative rate of normal neural tissues. The search for consistent or specific ultrastructural indicators of malignancy, or of neoplasia itself, has been a difficult task. n In this study, we found an increased incidence of cilia in many kinds of neoplasms. From our findings and from those of previous reports, 19,2~ we believe that the incidence of cilia may be an ultrastructural marker for cell proliferation, and that cell proliferation and the malignancy of the neoplasia can be correlated with the formation of cilia. Further,
G.G. Ganev: Cilia and centrioles in tumors the presence and incidence of free centrioles should also be examined, since cilia are absent during mitosis, and to increase the understanding of cell proliferation. Although the precise function of rudimentary cilia remains to be determined, 2~cilia formation seems to be involved in the process of cell division. We found no cilia in normal nerve cells, but it is possible that cilia are present in all tissues or cells, regardless of tissue maturity, and regardless of whether they are normal, inflammatory, or neoplastic. 2~ The incidence of cilia and centrioles in the cells of a tumor may be an ultrastructural marker of cell proliferation and, if used together with other methods (flow cytometry, thymidine labelling), determination of this incidence could help to establish the kinetic properties of human tumors.
Summary This study showed a higher incidence of cilia and centrioles in a variety of benign and malignant tumors compared to incidence in normal tissues. We believe this to be an ultrastructural manifestation of cell proliferation, but it may also be related to possible changes in the cell environment caused by tumor growth. Our findings suggest that an increased n u m b e r of cilia and/or centrioles may indicate malignancy. Nevertheless, ultrastructural details should not be considered diagnostically important until they are considered in the context of other changes, such as anaplasia, increased mitoses and chromosomal abnormalities, present in the tissue.
Acknowledgments. The
author was sponsored by the Japanese Ministry of Education and would like to thank the staff of the Departments of Orthopaedic Surgery and Pathology of Teikyo University (especially Prof. Akio Tateishi, Prof. Junji Shiga, Prof. Jo Sakakibara, Prof. Tohgo Ohno, and Dr. Pyoyun Park) for their support.
References 1. Armengot M, Escribano A, Carda C, et al. Clinical and ultrastructural correlations in nasal mucociliary function observed in children with recurrent airways infections. Int J Pediatr Otorhinolaryngol 1995;32:143-51 2. Bray D. Cell movements. New York: Garland, 1992:266-90. 3. Bubel A, Fitzsimmons C. Microstructure and function of cells. Chichester: Ellis Horwood, 1989:103. 4. Carson JL, Collier AM, Fernald GW, et al. Microtubular discontinuities as acquired ciliary defects in airway epithelium of patients with chronic respiratory diseases. Ultrastruct Pathol 1994;18:327-32. 5. Chan WY, Pak KH, Leung KM, et al. Gastric adenocarcinoma with ciliated tumor cells. Hum Pathoi 1993;24:1107-13.
G.G. G a n e v : Cilia a n d centrioles in t u m o r s 6. Dorfman HD. Bone cancers. Cancer 1995;75(Suppl 1):203-10. 7. Ferenczy A. The ultrastructural morphology of gynecologic neoplasms. Cancer 1976;38:463-86 8. Ghadially FN. Ultrastructural pathology of the cell and matrix. 2nd ed. London: Butterworths, 1982. 9. Gould PR, Henderson DW, Barter RA, et al. Cilia and ciliogenesis in endometrial adenocarcinomas. An ultrastructural analysis. Arch Pathol Lab Med 1986;110:326-30. 10. Hassan M, Subramanyan S. Ciliated renal tubular cells in crescentic glomerulonephritis. Ultrastruct Pathol 1995;19:201-3. 11. Henderson WH, Papadimitriou JM, Coleman M. Ultrastructural appearances of tumors. 2nd ed. Edinburgh: Churchill Livingstone, 1986:1 13. 12. Ho KL, Canccamo DV, Garcia JH. Intracytoplasmic lumina in ependymomas: An ultrastructural study. Ultrastruct Pathol 1994;18:371-80. 13. Ho PTC, Tucker RW. Centriole ciliation and cell cycle variability during G1 phase of BALB/c 3T3 cells. J Cell Physiol 1989; 139:398-406. 14. Katsumoto T, Higaki K, Ohno K, et al. The orientation of primary cilia during the wound response in 3YI cells. Biol Cell 1994;81:1721. 15. Lurie M, Rennert G, Goldenberg S, et al. Ciliary ultrastructure in primary ciliary dyskinesia and other chronic respiratory conditions: The relevance of microtubular abnormalities. Ultrastruct Pathol 1992;16:547-53. 16. Mierau GW, Agostini R, Beals TF, et al. The role of electron microscopy in evaluating ciliary dysfunction: Report of a workshop. Ultrastruct Pathol 1992;16:245-54. 17. Milhaud M, Pappas GD. Cilia formation in the adult cat brain after pargyline treatment. J Cell Biol 1968;37:600-9 18. Morinaga S, Tsumuraya M, Nakajima T, et al. Ciliated-cell adenocarcinoma of the pancreas. Acta Pathol Jpn 1986;36:190510.
145 19. Ohno T, Park P. An ultrastructural study of rudimentary cilia in the malignant and benign tumors of peripheral nerves. J Clin Electron Microsc 1983;16:203-9. 20. Park P, Manabe S, Ohno T. Incidence and ultrastructure of rudimentary cilia in benign and malignant peripheral nerve tumors. Ultrastruct Pathol 1988;12:407-18. 21. Park P, Yamamoto S, Kohmoto K, et al. Comparative effects of fixation methods using tannic acid on contrast of stained and unstained sections from Spurr-embedded plant leaves. Can J Bot 1982;60:1796-804. 22. Rayner CFJ, Rutman A, Dewar A, et al. Ciliary disorientation in patients with chronic upper respiratory tract inflammation. Am J Respir Crit Care Med 1995;151:800-4. 23. Sara A, Bruner JM, Mackay B. Ultrastructure of ependymoma. Ultrastruct Pathol 1994;18:33-42. 24. Schnepf E, Hausmann K, Herth W. The osmium tetroxidepotassium ferrocyanide (OsFeCN) staining technique for electron microscopy. A critical evaluation using ciliate~, algae, and higher plants. Histochemistry 1982;76:261-71. 25. Sorokin SP. Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J Cell Biol 1968;3:20%30. 26. Tashiro Y, Sueishi K, Nakao K. Nasal glioma: An immunohistochemical and ultrastructural study. Pathol Int 1995;45:393-8. 27. Torikata C, Kawai T, Nakayama M. Confronting cisternae and ciliated cells in malignant pleural mesothelioma: An ultrastructural study. Ultrastruct Pathol 1991;15:249-56. 28. Verra F, Escudier E, Lebargy F, et al. Ciliary abnormalities in bronchial epithelium of smokers, ex-smokers, and nonsmokers. A m J Respir Crit Care Med 1995;151:630-4.
~
lournalof
thopaedic Science TheJapanese Orthopaedic Association
Pathological significance of cilia and centrioles in bone and soft tissue, tumors GERSON GANDHI GANEV Teikyo University School of Medicine, Department of Orthopaedic Surgery, 2-11-I Kaga, Itabashi-ku, Tokyo 173, Japan
Abstract: The incidence and ultrastructure of rudimentary cilia and free centrioles were examined with a transmission electron microscope in 9481 cells from 74 bone and soft tissue tumors (benign and malignant) and from 11 samples of normal, histologically related tissues. The cilia were rudimentary (nonmotile), with a 9 + 0 axonemal pattern (occasionally 7 + 0 or 5 + 0), lacking the central tubules and two dynein arms of subfiber A, and with elongated shafts and basal structures consisting of two centrioles. Free centrioles appeared in all tissues studied, in general close to the Golgi apparatus. There was a significantly higher incidence of cilia and centrioles in the malignant tumors, compared to the incidence in benign tumors and normal tissues, and there was an inverse correlation between the incidence of cilia and centrioles in malignant tumors. Centrioles were not observed in ciliated cells, nor were cilia observed in cells undergoing mitosis. These findings suggest that neoplasia can lead to an increase in the incidence of cilia and centrioles, and that the increase correlates with the malignancy of a tumor. Consideration of the incidence of cilia and centrioles probably gives a better idea of cell proliferation than consideration of the incidence of cilia only. Key words: bone and soft tissue tumors, ultrastructure, cilia
Introduction The presence of cilia has been reported in a variety of tissues throughout the animal kingdom. Cilia in animal tissues can be motile or immotile. The latter are also
Offprint requests to: G.G. G a n e v Received for publication on Aug. 2, 1996; accepted on Dec. 10, 1996
called oligocilia, primary cilia, rudimentary cilia, or solitary cilia. Motile and immotile cilia differ in their m o d e of morphogenesis, ultrastructure, and function. Motile cilia have a "9 + 2" axonemal pattern, which consists of nine doublet microtubules in the peripheral area and two singlet microtubules in the central area. The doublet microtubules are c o m p o s e d of subfiber A, with two dynein arms involving ATPase, and subfiber B. Motile cilia can undergo pathological ultrastructural changes in a variety of diseases, 1,4,1~ though some changes may be reversible. 1,z2 C o m p a r e d with motile cilia, rudimentary cilia were r e p o r t e d to lack the central pair of microtubules, resulting in a "9 + 0" structure. Further, Park et al. 2~reported that two dynein arms of subfiber A were absent in r u d i m e n t a r y cilia of Schwann cells of a malignant schwannoma, and that rudimentary cilia were immotile because of the absence of the dynein arms. Most vertebrate cells produce apparently nonfunctional, rudimentary cilia, which have b e e n observed in a variety of tissues, such as smooth muscle, brain, liver, lungs, spleen, and testis? R u d i m e n t a r y cilia also occur in carcinomas 5 and sarc o m a s ? Park et al? ~ reported a higher incidence of cilia in malignant peripheral nerve tumors than in benign tumors. They concluded that this feature could possibly be an ultrastructural m a r k e r for cell proliferation in tumors. They also noted that cilia a p p e a r e d to be closely associated with cell division in nerve tumors, and that the incidence of cilia was inversely correlated with that of free centrioles. However, the incidence and relationship of cilia and free centrioles in other types of tumors have not yet been studied. In this investigation, we investigated the incidence of cilia and free centrioles in bone, cartilage, and nerve tumors, as well as in normal tissues histologically related to the tumors. The purpose of this study was to establish the pathological significance of cilia and centrioles in t u m o r cells.
138
G.G. Ganev: Cilia and centrioles in tumors
Materials and m e t h o d s F r e s h tissues f r o m 8 o s t e o s a r c o m a s , 6 E w i n g ' s s a r c o m a s of bone, 9 chondrosarcomas, 9 malignant schwannomas, 14 e n c h o n d r o m a s , 20 b e n i g n p e r i p h e r a l n e r v e t u m o r s (neurofibroma and schwannoma), and 8 miscellaneous t u m o r s w e r e o b t a i n e d at surgery. N o r m a l tissues (3 s a m p l e s o f histologically n o r m a l sciatic n e r v e , 1 o f nor-
m a l n e r v e in skin tissue, an d 8 o f n o r m a l c a r t i l a g e ) w e r e o b t a i n e d f r o m a m p u t a t e d limbs. A l l tissues w e r e cut into small p i e c e s with a r a z o r blade. T h e m a t e r i a l s w e r e t h e n i m m e r s e d in 2.5% g l u t a r a l d e h y d e and 2 % p a r a f o r m a l d e h y d e , b u f f e r e d with 0 . 1 M c a c o d y l a t e b u f f e r at p H 7.4, an d w e r e k e p t o v e r n i g h t at 4~ A f t e r wards, t h e y w e r e r i n s e d s e v e r a l t i m es with t he s a m e buffer, an d t h e n e x p o s e d to 1% b u f f e r e d o s m i u m
Table 1. Tumors and related tissues examined No. of cases examined
Cell type
No. of cells examined
Osteosarcoma Osteosarcoma Osteosarcoma Ewing's sarcoma of bone Chondrosarcoma Enchondroma Normal cartilage (growth plate) Malignant schwannoma Schwannoma Neurofibroma Normal nerve Primitive neuroectodermal tumor Liposarcoma Intramuscular myxoma Malignant hemangiopericytoma Clear cell sarcoma Chordoid sarcoma Epithelioid sarcoma
2 5 1 6 9 14 8 9 8 12 3 1 2 1 1 1 1 1
Osteoblast Chondroblast Fibroblast Tumor cell Chondroblast Chondroblast Chondrocyte Schwann cell Schwann cell Schwann cell Schwann cell Tumor cell Lipoblastic cell Tumor cell Endothelial cell Tumor cell Tumor cell Tumor cell
114 449 99 452 1007 1445 605 2107 970 868 557 77 180 67 200 100 98 86
Total
85
Tissues
9481
Table 2. Incidence a of cilia and centrioles and statistical evaluation in tumors and related tissues Tissues
No. of cases
Osteosarcoma Ewing's sarcoma of bone Chondrosarcoma Enchondroma Normal chondrocytes from cartilage (growth plate) Malignant schwannoma Benign nerve tumors Normal nerve Miscellaneous tumors
1:4 1:7 1:16 4:7 4:16 7:16 7:10 7:13 10:13 16:19
NS P < 0.01 NS P < 0.01 NS NS P<0.01 P<0.01 NS P<0.01
Percentage of cells with cilia
8 6 9 14 8
1. 4. 7. 10. 13.
9 20 3 8
16. 5.9 _+ 4.2 19. 1.1 _+ 1.2 0 5.8
2:5 P < 0.05 3:6 NS 2:8 P < 0.01 3:9 P < 0.05 2:17 P < 0 . 0 5 3:18 NS 5:8 NS 6:9 P < 0.01 5:17 NS 6:18 P<0.05 8:17 NS 9:18 NS 8:11 P<0.01 9:12 P<0.01 8:14 NS 9:15 P<0.01 11:14 NS 12:15 NS 17:20 P<0.01 18:21 P<0.01 17:22 P < 0 . 0 5 18:23 P<0.05 20:22 NS 21:23 P < 0.05 NS, Non-significant. Comparison was done with the Wilcoxon test. aMean values _+ SD (for miscellaneous tumors, mean value only)
2.5_+ 1.2 3.0_+2.0 8.1 _+ 3.3 2.0 _+ 1.6 2.4 _+ 2.5
Percentage of cells with centrioles 2. 5. 8. 11. 14.
4:10 7:19 10:16 8:19
Percentage of cells with cilia and with centrioles
7 . 2 + 1.9 4.8_+2.3 4.1 _+ 3.6 1.6 _+ 0.9 2.4 _+ 1.9
3. 9.7 + 2 . 0 6. 7.7_+2.2 9. 12.2 _+ 2.2 12. 3.6 _+ 1.7 15. 4.8 _+ 2.6
17. 4.6 _+ 2.6 20. 1.6 _+ 1.4 22. 0.1 _+ 0.2 4.6
18. 10.5 + 1.9 21. 2.6 _+ 1.8 23. 0.1 +_ 0.2 10.4
NS P<0.01 P<0.01 NS
G.G. Ganev: Cilia and centrioles in tumors tetroxide for 2h. Some specimens were fixed by the tetra-fixation method, using glutaraldehyde, tannic acid, and osmium tetroxide. 21Some tissues from bone tumors were fixed with a mixture of 1% glutaraldehyde and 4% paraformaldehyde in 0.1M cacodylate buffer for 2448h at room temperature. After prefixation, the bone tumors were treated with 5% ethylene diamine tetraacetic acid ( E D T A ) for 1-2 weeks at room temperature, and then postfixed with osmium tetroxide. Some cartilaginous tissues (such as enchondroma and normal cartilage) and some l~erve tumors were fixed with a mixture of osmium tetroxide and potassium ferrocyanide, according to the method described by Schnepf et al., 24 after aldehyde fixation. All specimens were dehydrated with ethanol and embedded in Epon 812, Quetol 812, and Spurr resin. Ultrathin sections cut from the resin blocks were stained with uranyl acetate and lead citrate. The stained sections were observed with J E O L (Tokyo, Japan) 100 S and 1200EX electron microscopes. Electron microscopic negatives were taken at random at x2000 magnification from multiple areas of many sections from approximately 800 blocks. The negatives were enlarged at x 1 0 0 0 0 - x 1 8 0 0 0 and printed on large photographic paper, and every cell in the prints was studied individually, using a loupe to identify cilia and centrioles. Before determining the incidence of cilia we examined the cell type in each sample, and only cells that characterized the tumor (for example, osteoblasts in a osteoblastic type osteosarcoma) were entered in this:study. The cells and tissues studied are summarized in Table 1. For statistical analysis, the mean percentage and standard deviation were calculated for the incidence of cilia and centrioles in each group of benign tumors, malignant tumors, and normal cells. The values were then estimated with the Wilcoxon test; P < 0.05 was considered a significant difference. Overall statistical results are summarized in Table 2. Benign nerve tumors (schwannoma and neurofibroma) were statistically analyzed as one group. The incidence of cilia and centrioles in the miscellaneous tumors was examined, but not included in the general statistical analysis.
139 quently, in longitudinal profile (Figs. 6, 7). Most cilia were seen at the cell periphery, but some were situated deep in the cytoplasm in close association with invaginated cell membranes (Fig. 8). The ultrastructure of rudimentary cilia was similar in benign and malignant tumors, and also in normal cells, such as normal chondrocytes. The cilia consisted of long shafts with a 9 + 0 axonemal pattern (Fig. 2), but other patterns also appeared, such as 8 + 0, 7 + 0, 6 + 0, and 5 + 0 (Fig. 3). Each of the nine peripheral doublets consisted of subfibers A and B. The two dynein arms of subfiber A, normally present in motile cilia, were absent in the cilia of osteosarcoma, chondrosarcoma, malignant schwanoma, clear cell sarcoma, and normal chondrocytes (Fig. 2). At the base of the cilium, a basal body, consisting of two centrioles, was present, attached to the end of the axonemes (Figs. 7, 9). The transitional fibers emerging from the basal body were connected with cell mem-
Fig. 1. In malignant schwannoma, four rudimentary cilia were found in the extracellular space in the vicinity of a Schwann cell membrane
Results
Ultrastructural findings Rudimentary cilia usually occurred singly in tumor cells and the related normal cells, except for Schwann cells of normal nerve, where cilia were not found. In some Schwann cells of malignant schwannoma, several cilia per cell were seen (Fig. 1). Cilia were sometimes seen in transverse and oblique profiles (Figs. 1-5) or, less fre-
Fig. 2. Transverse profile of a rudimentary cilium in an osteoblast of osteosarcoma. The cilium has a 9 + 0 axonemal pattern of microtubules. Note that two dynein arms are absent in subfiber A of the cilium (arrowhead)
140
Fig. 3. Rudimentary cilium with a 5 + 0 axonemal pattern of
G.G. Ganev: Cilia and centrioles in tumors branes. The basal body contained well developed adnexa, such as triangular basal feet and some long striated rootlets (Figs. 8, 9). Two basal feet could be seen in the side walls of a basal body (Figs. 7, 9). A free centriole was usually observed in tumor cells and in cells of normal tissues, in general near the Golgi apparatus (Fig. 10). Cilia and free centrioles were not observed in the same cell. When cilia were found in the cells examined, these ciliated cells contained no centrioles, and when centrioles were found in the cells, no cilia were present. Moreover, no cilia were observed in cells undergoing mitosis, although centrioles usually appeared in the mitotic cells.
a Schwann cell in malignant schwannoma
Fig. 4. Ciliary shaft (arrowhead) in an osteoblast of osteosarcoma
Fig. 5. Rudimentary cilium chondroblast in enchondroma
of
a
G.G. Ganev: Cilia and centrioles in tumors
141
Statistical analysis Incidence of cilia. Chondrosarcoma and malignant
Fig. 6. Ciliated chondroblast in chondrosarcoma
schwannoma were the tumor groups showing the highest incidence of cilia, and osteosarcoma and Ewing's sarcoma showed the next highest incidence (Table 2; Fig. 11). The difference between chondrosarcoma and osteosarcoma in incidence of cilia was significant and the difference between chondrosarcoma and Ewing's sarcoma was also significant, although there was no significant difference between malignant schwannoma and osteosarcoma, or between malignant schwannoma and Ewing's sarcoma (Table 2). Compared to the benign tumors, the malignant tumors usually had a higher incidence of cilia (Table 2; Fig. 11); there were significant differences between
Fig. 7. Typical rudimentary cilium in clear cell sarcoma. The cilium has basal bodies, a long elongated ciliary shaft, and triangular basal feet structures
Fig. 8. Ciliary shaft (oblique profle) seen in a Schwann cell in malignant schwannoma. The cytoplasm in the vicinity of the ciliary base involves a rootlet structure (arrowheads)
Fig. 9. Basal bodies in a Schwann cell of a neurofibroma, showing two triangular basal feet structures (arrowheads)
142
G.G. Ganev: Cilia and centrioles in tumors
Fig. 10. Free centriole in an osteoblast of osteosarcoma
9 8 7 6 5 4 3 2 1 0 NN
NC
EC
SW
NF
MS
CS
OS
EW
Fig. 11. Average incidence of cilia in the tissues studied. NN, Normal nerve; NC, normal chondrocyte; EC, enchondroma; SW, Schwannoma; NF, neurofibroma; MS, malignant schwannoma; CS, chondrosarcoma; OS, osteosarcoma; EW, Ewing's sarcoma
malignant schwannoma and benign nerve tumors, between chondrosarcoma and enchondroma, between chondrosarcoma and benign nerve tumors, and between malignant schwannoma and enchondroma. The benign nerve tumors had a higher incidence of cilia than the normal nerves, whereas enchondromas and normal nerves showed no difference in incidence. Normal chondrocytes showed a higher incidence of cilia than enchondromas (Table 2); this might have been because the cartilage samples examined were from the growth plate, where cell proliferation is higher than in normal cartilage. In the normal tissues examined, normal chondrocytes often had cilia, as did the tumor cells of enchondroma, but Schwann cells of normal nerve did not have any cilia.
Incidence of free centrioles. Among malignant tumors, osteosarcoma and Ewing's sarcoma had the highest incidence of free centrioles, followed by malignant schwannoma and chondrosarcoma; osteosarcoma showed an especially high incidence, and the difference between this tumor and all other types of malignant tumors was significant (Table 2, Fig. 12). Differences among Ewing's sarcoma, chondrosarcoma, and malignant schwanoma were not significant. Enchondroma and benign nerve tumors showed a low incidence of centrioles. Normal cartilage showed a higher incidence than benign tumors, but the cartilage tissue studied here was from areas involving the growth plate, which differs from articular cartilage. Normal nerve had the lowest incidence of centrioles. Overall, malignant tumors consistently showed a higher incidence of centrioles than benign tumors and normal tissues (Table 2, Fig. 12): the difference was significant for both benign tumors and normal tissues. However, the difference in incidence of free centrioles between benign tumors and normal tissues was not significant (Table 2). Combined incidence of cells with cilia and cells with free centrioles. The combined incidence of ciliated cells and cells with free centrioles showed three apparently distinct tissue groups: malignant tumors, which had a high incidence (probably due to the highest proliferative activity), benign tumors and the cartilage growth plate, which had a moderate incidence, and normal tissue, which had a low incidence (Table 2, Fig. 13). Functionally, the centriole is the "common component" shared by cilia and free centrioles (since the ciliary basal body is formed by the centrioles), so the combined incidence of both structures corresponds to the incidence of
G.G. Ganev: Cilia and centrioles in tumors
143
centrioles that arise from the basal bodies of cilia and those that arise from the free centrioles of the cytoplasm. Miscellaneous tumors, which consisted mostly of sarcomas, showed a high incidence of both cilia and centrioles (Table 2). In malignant tumors, we found a negative correlation coefficient between the incidence of cilia and centrioles; this indicates that a high incidence of cilia in a malignant tumor was associated with a low incidence of centrioles, and a high incidence of centrioles was associated with a low incidence of cilia. This was not observed in benign tumors or in normal cells.
Ciliated cells have been documented in many neoplasms, including peripheral nerve tumors; ~9,2~gliomas; 26 ependymomas; 23 adenocarcinomas of the stomach, uterus, and pancreas; 5,7,~8 endometrial carcinoma; 9 and mesothelioma? 7 Further, ciliary abnormalities have been described in inflammatory conditions, including chronic respiratory diseases 4~22,28 and glomerulonephritis. 1~ The opportunity to encounter cilia and free centrioles in thin sections is rare, and analysis of the incidence depends on observations confined to small areas of specimens. Therefore, in this study, we attempted to observe more samples from each subject, containing as many ceils as possible. Additionally, we examined some tissues in which the presence of cilia, to the best of our knowledge, has not been documented before, such as bone sarcomas and enchondroma. Cilia found in tumors are mostly of the rudimentary (immotile) type, with a 9 + 0 axonemal pattern? ,~9,2~In this study, we found variations of the traditional pattern of axonemes (9 + 0) in rudimentary cilia, including 8 + 0, 7 + 0, 6 + 0, and 5 + 0 patterns; this may indicate that the cilia were cut at different heights in the ciliary shaft?
We also found cilia in all tissues studied, except in Schwann cells of normal nerve. We believe that cilia are distributed in all tissues or cells, including normal, inflammatory, and neoplastic. The occurrence of rudimentary cilia has been explained in different ways. Torikata et al. 27 reported ciliated cells in a malignant pleural mesothelioma and suggested that mesothelial cells could be changed into ciliated cells in the process of tumorigenesis. Bray 2 suggested that the cell "program" for making a cilium could be activated in many different situations in which an extension of the cell surface was needed, even if movement was not required. Park et al., 2~ describing an increased incidence of cilia in peripheral nerve tumors, emphasised the inverse correlation betv)een cilia formation and mitosis, and the absence of cilia in mitotic cells, despite the presence of cilia in highly proliferative tissues. Milhaud and Papas 17 induced ciliogenesis in cat brain cells with pargyline (a m o n o a m i n e oxidase inhibitor) and suggested that centriolar production without subsequent mitosis may lead to the production of cilia. The relationship between cilia and centrioles, and between centrioles and cell division, is the key to clarifying the role of cilia and centrioles in tumor cells: cilia arise from basal bodies, and basal bodies from centrioles. 2 Centrioles are part of the cell centrosome, which is the major microtubule-organizing center in most animal cells, involved in cell m o v e m e n t and cell division. 2 Rudimentary cilia are formed by one of the interphase centrioles, and the occasional presence of aberrant intracytoplasmic cilia (common in ependymomas) has been interpreted as a failure in the migration of the basal body to the p r o p e r apical surface of the cell. 12,15Centriole ciliation has been shown to precede D N A synthesis by 5--6h; 13 accordingly, Katsumoto et al. TM have suggested that the organization of cilia plays an important role in controlling the cell's commitment to initiation of D N A synthesis.
JCentrioles% J
I Cilia+Centrioles%
Discussion
I
20
8765-
10
432
10-
0 NN
NC
EC
SW
NF
MS
CS
06
EW
Fig. 12. Average incidence of free centrioles in the tissues studied. For abbreviations, see Fig. 11
NN
NC
~
EC
SW
NF
MS
CS
OS
EW
Fig. 13. Average incidence of ciliate cells plus cells with free centrioles. For abbreviations, see Fig. 11
144 We found a higher incidence of cilia in malignant than in benign tumors; and this can be interpreted as a sign of high cell proliferation in malignant tumors; the association of cilia with poor tumor differentiation was not clear in this study, although it has been reported before. 19Cilia and centrioles were not found in the same cell in our study, and we found a negative correlation between the incidence of cilia and centrioles in malignant tumors. Cilia and free centrioles appeared at different stages of the cell cycle (centrioles being "consumed" in the formation of the ciliary basal body), and the relationship between cilia and centrioles was more clearly expressed in malignant tumors, where cell proliferation was probably higher. Osteosarcoma is the most c o m m o n malignant bone tumor, followed by chondrosarcoma and Ewing's sarcoma? The incidence of cilia was not high in osteosarcoma and Ewing's sarcoma of bone. On the other hand, these tumors had the highest incidence of centrioles of all tissues studied. This may indicate a high mitotic rate in these tumors, which could explain both findings i.e., the low incidence of cilia and the high incidence of centrioles (cilia are not found in mitotic cells). The difference in the cilia-centriole profile between osteosarcoma and chondrosarcoma (which had the highest incidence of cilia among all tissues studied) may help to explain why sensibility to chemotherapy is different in these tumors, with osteosarcoma probably having a larger growth fraction (percentage of proliferating cells in a tumor). Analysis of the incidence of both cilia and centrioles may provide a more precise indication of tumor proliferation, since both structures seem to be involved in cell division at different stages of the cell cycle. We found that normal tissue with high proliferation (cartilage growth plate) had a high incidence of centrioles, even higher than that seen in benign tumors (Table 2, Fig. 12). Cell kinetics would seem to differ greatly between benign (Schwannoma, neurofibroma) and malignant nerve tumors (malignant schwannoma), since differences in the incidence of both cilia and centrioles were highly significant ( P < 0.01). Differences between benign nerve tumors and normal nerve were not as significant, and it seems that benign tumors retain most of the normal, low proliferative rate of normal neural tissues. The search for consistent or specific ultrastructural indicators of malignancy, or of neoplasia itself, has been a difficult task. n In this study, we found an increased incidence of cilia in many kinds of neoplasms. From our findings and from those of previous reports, 19,2~ we believe that the incidence of cilia may be an ultrastructural marker for cell proliferation, and that cell proliferation and the malignancy of the neoplasia can be correlated with the formation of cilia. Further,
G.G. Ganev: Cilia and centrioles in tumors the presence and incidence of free centrioles should also be examined, since cilia are absent during mitosis, and to increase the understanding of cell proliferation. Although the precise function of rudimentary cilia remains to be determined, 2~cilia formation seems to be involved in the process of cell division. We found no cilia in normal nerve cells, but it is possible that cilia are present in all tissues or cells, regardless of tissue maturity, and regardless of whether they are normal, inflammatory, or neoplastic. 2~ The incidence of cilia and centrioles in the cells of a tumor may be an ultrastructural marker of cell proliferation and, if used together with other methods (flow cytometry, thymidine labelling), determination of this incidence could help to establish the kinetic properties of human tumors.
Summary This study showed a higher incidence of cilia and centrioles in a variety of benign and malignant tumors compared to incidence in normal tissues. We believe this to be an ultrastructural manifestation of cell proliferation, but it may also be related to possible changes in the cell environment caused by tumor growth. Our findings suggest that an increased n u m b e r of cilia and/or centrioles may indicate malignancy. Nevertheless, ultrastructural details should not be considered diagnostically important until they are considered in the context of other changes, such as anaplasia, increased mitoses and chromosomal abnormalities, present in the tissue.
Acknowledgments. The
author was sponsored by the Japanese Ministry of Education and would like to thank the staff of the Departments of Orthopaedic Surgery and Pathology of Teikyo University (especially Prof. Akio Tateishi, Prof. Junji Shiga, Prof. Jo Sakakibara, Prof. Tohgo Ohno, and Dr. Pyoyun Park) for their support.
References 1. Armengot M, Escribano A, Carda C, et al. Clinical and ultrastructural correlations in nasal mucociliary function observed in children with recurrent airways infections. Int J Pediatr Otorhinolaryngol 1995;32:143-51 2. Bray D. Cell movements. New York: Garland, 1992:266-90. 3. Bubel A, Fitzsimmons C. Microstructure and function of cells. Chichester: Ellis Horwood, 1989:103. 4. Carson JL, Collier AM, Fernald GW, et al. Microtubular discontinuities as acquired ciliary defects in airway epithelium of patients with chronic respiratory diseases. Ultrastruct Pathol 1994;18:327-32. 5. Chan WY, Pak KH, Leung KM, et al. Gastric adenocarcinoma with ciliated tumor cells. Hum Pathoi 1993;24:1107-13.
G.G. G a n e v : Cilia a n d centrioles in t u m o r s 6. Dorfman HD. Bone cancers. Cancer 1995;75(Suppl 1):203-10. 7. Ferenczy A. The ultrastructural morphology of gynecologic neoplasms. Cancer 1976;38:463-86 8. Ghadially FN. Ultrastructural pathology of the cell and matrix. 2nd ed. London: Butterworths, 1982. 9. Gould PR, Henderson DW, Barter RA, et al. Cilia and ciliogenesis in endometrial adenocarcinomas. An ultrastructural analysis. Arch Pathol Lab Med 1986;110:326-30. 10. Hassan M, Subramanyan S. Ciliated renal tubular cells in crescentic glomerulonephritis. Ultrastruct Pathol 1995;19:201-3. 11. Henderson WH, Papadimitriou JM, Coleman M. Ultrastructural appearances of tumors. 2nd ed. Edinburgh: Churchill Livingstone, 1986:1 13. 12. Ho KL, Canccamo DV, Garcia JH. Intracytoplasmic lumina in ependymomas: An ultrastructural study. Ultrastruct Pathol 1994;18:371-80. 13. Ho PTC, Tucker RW. Centriole ciliation and cell cycle variability during G1 phase of BALB/c 3T3 cells. J Cell Physiol 1989; 139:398-406. 14. Katsumoto T, Higaki K, Ohno K, et al. The orientation of primary cilia during the wound response in 3YI cells. Biol Cell 1994;81:1721. 15. Lurie M, Rennert G, Goldenberg S, et al. Ciliary ultrastructure in primary ciliary dyskinesia and other chronic respiratory conditions: The relevance of microtubular abnormalities. Ultrastruct Pathol 1992;16:547-53. 16. Mierau GW, Agostini R, Beals TF, et al. The role of electron microscopy in evaluating ciliary dysfunction: Report of a workshop. Ultrastruct Pathol 1992;16:245-54. 17. Milhaud M, Pappas GD. Cilia formation in the adult cat brain after pargyline treatment. J Cell Biol 1968;37:600-9 18. Morinaga S, Tsumuraya M, Nakajima T, et al. Ciliated-cell adenocarcinoma of the pancreas. Acta Pathol Jpn 1986;36:190510.
145 19. Ohno T, Park P. An ultrastructural study of rudimentary cilia in the malignant and benign tumors of peripheral nerves. J Clin Electron Microsc 1983;16:203-9. 20. Park P, Manabe S, Ohno T. Incidence and ultrastructure of rudimentary cilia in benign and malignant peripheral nerve tumors. Ultrastruct Pathol 1988;12:407-18. 21. Park P, Yamamoto S, Kohmoto K, et al. Comparative effects of fixation methods using tannic acid on contrast of stained and unstained sections from Spurr-embedded plant leaves. Can J Bot 1982;60:1796-804. 22. Rayner CFJ, Rutman A, Dewar A, et al. Ciliary disorientation in patients with chronic upper respiratory tract inflammation. Am J Respir Crit Care Med 1995;151:800-4. 23. Sara A, Bruner JM, Mackay B. Ultrastructure of ependymoma. Ultrastruct Pathol 1994;18:33-42. 24. Schnepf E, Hausmann K, Herth W. The osmium tetroxidepotassium ferrocyanide (OsFeCN) staining technique for electron microscopy. A critical evaluation using ciliate~, algae, and higher plants. Histochemistry 1982;76:261-71. 25. Sorokin SP. Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J Cell Biol 1968;3:20%30. 26. Tashiro Y, Sueishi K, Nakao K. Nasal glioma: An immunohistochemical and ultrastructural study. Pathol Int 1995;45:393-8. 27. Torikata C, Kawai T, Nakayama M. Confronting cisternae and ciliated cells in malignant pleural mesothelioma: An ultrastructural study. Ultrastruct Pathol 1991;15:249-56. 28. Verra F, Escudier E, Lebargy F, et al. Ciliary abnormalities in bronchial epithelium of smokers, ex-smokers, and nonsmokers. A m J Respir Crit Care Med 1995;151:630-4.