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Electron microscopy of human choriocarcinoma transplanted into hamster liver
FETUS AND NEWBORN Electron microscopy of human choriocarcinoma transplanted into hamster liver ]. FALCK LARSEN, M.D. ROBERT L. EHRMANN, M.D. FRANZ BIERRING, M.D. Aarhus and Odense, Denmark, and Brookline, Massachusetts Human choriocarcinoma was transplanted into the liver of 26 hamsters in order to study the ultrastructure of tumor and junction zone. Two types of tumor cells were observed. The first type resembled the cytotrophoblast of the normal placenta, but the cells were larger and contained large irregular mitochondria with few cristae. The second type resembled the normal syncytiotrophoblast, and some exhibited phagocytotic activity. Liver nuclei and cytoplasmic components of liver cells were observed in some of the trophoblastic cells. Liver cells adjacent to tumor showed proliferation of the agranular reticulum and the presence of polyribosomes resembling those of the tumor cells. Desmosomes (submicroscopic bindings) were observed con•necting tumor cells to liver cells. On the basis of present and past observations, a comparison is made between invasion by choriocarcinoma and implantation by the normal fertilized egg.
I M P L A N T A T I o N of the ovum has been studied intensively during the last few years (see reviews by Eckstein/ Ferin and Gaudefroy/0 Larsen,17 and Wolstenholme and O'Connor 31 ). These investigations have included studies of the reactions between trophoblast and invaded tissue at the ultrastructural level. 6 • 15 • 22 Electron microscopic
studies of the early human trophoblast during implantation have not yet been possible because of problems in obtaining the material. The earliest human trophoblast studied with the electron microscope is from a four-somite embryo. 19 Hertig and ManselP 2 draw attention to the resemblance between choriocarcinoma and trophoblast of the first week after implantation. Due to the lack of early human material, a study on the ultrastructure of choriocarcinoma may give information which will be useful in the understanding of trophoblastic invasion in man. Choriocarcinoma, however, is a rare tumor, and few investigators have been able to present ultrastructural observations on tumor taken directly from patients. 14 • 16 • 28 Hertz13 has been able to carry choriocarcmoma by serial passage in cheek pouches
From the Division of Electron Microscopy, University of Aarhus; The Parkway Division of The Boston Hospital for Women, Brookline; and the Department of Anatomy, University of Odense. This investigation was supported by United States Pubiic Health Service Grants No. CA 05905, HD 01261, HD 02493, and RF-44, The Lalor Foundation, The Association for the Aid of Crippled Children, and The Danish Anti-Cancer League.
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of hamsters, and this method made the tumor available for biologic studies. The ultrastructure of choriocarcinoma implanted into the cheek pouch was studied by vvynn and Davies/10 with special regard to endocrine implications. It is difficult to obtain well preserved tissue for electron microscopy from the cheek-pouch tumor and adjacent tissue, possibly due to the intensive destruction, or the abundance of blood which blocks fixation. Ehrmann and Gliserman 3 transplanted this tumor not only into cheek pouches but also into liver, brain, and other organs in hanlsters, and studied the junction zone with tht' light microscope. In the present investigation, the junction zone was studied with the electron microscope. Materials and methods
Experimentai series of hamsters.
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5 mm. x 2 rnm. slivers of the Green choriocarcinoma (obtained froin Dr. Roy Hertz and carried by hamster cheek pouch transfer approximately every 10 days since February, 1962) were inoculated through an abdominal incision into the livers of 26 young female 80 to 100 gram Syrian hamsters, using a Lundy-Irving caudal needle. Eighteen of these grew suitable intrahepatic turflors which provided enough tumor-liver junctional tissue for the preparation of 125 Epon bloch Although cortisone was not used for the routine cheek pouch passage of the tumor, it was necessary for the maintenance of intrahepatic tumor growth. Each experimental hamster was given an intradermal injection twice weekly, of 0.05 ml. cortisone (Cortone i (50 mg. per milliliter) . The animals \vere
Table I. Experimental series of hams ten. No. of animals
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Lung metastases Ill I animal Lung metastases m I animal Lung metastases Ill 1 animal Lung metastases tn :l animals Lung metastases m 1 animal
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killed 14, 16, 17, 18, or 20 days after inoculation with tumor. The experiments are summarized in Table I. The animals were anesthetized with Nembutal and biopsies from representative areas of the junction zone ·between liver and tumor were cut down to blocks measuring approximately 1 to 2 mm.,a fixed in 1 per cent osmic acid in S-collidene buffer, 2 dehydrated in methyl alcohol and embedded in Epon.~" Sections were cut at 1 p. and stained with 1 per cent toluidine blue for light microscopy, and ultrathin sections were cut on LKB- and Porter-Blum ultramicrotomes. The sections were stained with lead citrate 27 or uranyl acetate. £!1 Electron micrographs were made with JEM-6C and Phillips-100 electron microscopes. Larger pieces of the junction zone were fixed in Bouin's solution, embedded in paraffin, and stained with hematoxylin and eosin for ordinary light In icroscopy.
Fig. 1. Junction zone parallel to the liver sinusoids. The line separating ~e liver cells (L) from trophoblast (T) is very regular. The syncytial type of cell (S) appears darker with this stain. The large tumor blood space is lined with trophoblasts. (x120.) Fig. 2. Trophoblastic cells (T) invading the liver sinU$oids. The trophoblasts contain dark corpuBcles, some of which are phagocytosed red blood cells. (x240.) Fiv_ 3. Trnnhnhla•to inv.adin.,. liver sinusoids. In some of the sinusoids the invasion takes place i~oth~- sJ>;.~;-~£-Di~~e--bt;t;~;n th~ llve~-cells (L) and the Kupffer cells (K). (x300.) Fig. 4. Portion of tumor with syncytial elements (S) and cytotrophoblast (C). Some of the blood spaces are lined with tumor tells (B), others with endothelium (V). (x120.)
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Transplanted choriocarcinoma
Figs. 1-8. Light mic rogra phs. Epon embedded. T oluidine blue stained.
Figs. 1-4. For legends see opposite page.
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Control series of hamsters. Fifty-one yourl~.! female hamsters ( 80 to 100 ,grams) were used in four different control groups. To evaluate the changes found in the liver cells of the junction zone, it was necessary to have tissue from livers of (a) normal hamsters--12 animals; (b) hamsters treated with cortisone-13 animals; (c) hamsters with choriocarcinoma in cheek pouches-12 animals ; and (d) hamsters with choriocarcinoma in cheek pouches and treated with cortisone14 animals. The control animals had the same were anesthetized the same way, and the tissues were obtained, fixed, and embedded using the same methods. To determine whether contact with liver tissue had any specific observable effect on the implanted tumor, an electron microscopic comparison was made with cheek pouch tumor from hamsters after cortisone (control group (d) -see above) and from hamster~ which received no cortisont>.
Results The tumor ranged from 1 to 2.5 em. in diameter and was dark red m color, often with central hemorrhage and necrosis. The trophoblastic tissue was found as a 0.5 to 1 mm. spherical shell surrounding the necrosts. Light microscopy of l,u Epon-embedded sections. The tumor possessed the light microscopic features of choriocarcinoma, being composed of anastomosing cords of cytotrophoblast n1ixed with syncytiotrophoblast. Blood sinusoids lined with trophoblastic cells as well as true vessels lined with endothelium were observed. In some limited areas collagen
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was found. b11t for tbl' mo;,t iKtrt the tUJnnr tissue contained no mesencbyuw. Different types of tumor cells wt·re found. In Soine areas the trophoblast consisted of mononuclear, spindle-shaped cells (Fig~. ,.) and 6\ while other an· as were dominated by larger cells or syncytial masses (Figs. 4 and 8) . Most of the trophoblastic cells were Je,s heavily stained than the liver cells, probably because of the abundance of ribosomes and glycogen in the latter cells. However, some of the trophoblastic cells were intensively ~:tained (Fig. 4) .· and these cells were. found to be rich in endoplasmic reticulum when examined under the electron microscope (Figs. 15 and 16\. Some of the cells had a strange "foamy" cytoplasm (Figs. 6 and 7 i caused by large vacuoles, whereas other cells contained one large vacuole in tbe center of the cytoplasm displacing the nucleus (Figs. 5 and 61. The size of the tumor cells varied greatly, ranging from cytotrophoblastic cells measuring about 15 ,u to multinucleated giant cells (syncytial masses) almost 1 mm. in diameter (Fig. 8). The nuclei were very large (some up to 60 ~~ in diameter), irregular in shape, and had large conspicuous nucleoli (Figs. 2 through 81. Many mitoses were found. (Fig. 6.1 . The junction zone between the tumor and liver was found to be very straight in most sections (Fig·. 1 ) . The borderline was especially regular in areas where the direction of invasion was perpendicuiar to that of the liver sinusoids (Fig. 1). In areas where invasion took place paralle-l to the liver sinusoids, the trophoblast seemed to invade the space of Disse (Figs. 2 and 3) between the liver cells and the sinusoid cells.
Fig. 5. Portion of tumor with cytotrophoblasts (C) and lacunae lined by trophoblasts (LA). Syncytial areas (S) are also found. M: Mitosis. (x300.) Fig. 6. Portion of tumor with "foamy" cell type (F), cytotrophoblasts (C), lacunae (LA), and many mitoses (M). (x300.) Fig. 7. Portion of tumor with "foamy" cells and blood vessel (B) lined with trophoblast (T). (x420.) Fig. 8. Syncytial mass of tumor (S) with numerous large nuclei which have large nucleoli and very irregular nuclear membranes. ( x240.)
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Figs. 5-8. For legends see opposite page.
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In many cases, single li\·er· cells or grou 1>S of two or three cells were found so deep in the tumor that their presence could not lw explained by tangential sectioning of an irregular borderline. These liver cells looked normal in most of the sections, but in some the cytoplasm appeared more basophilic than normal. Occasionally, degenerating fragments of probable liver cells could be seen within the tumor cytoplasm. Electron microscopy. Two different types of tumor cells were found. The most common type resembled the cytotrophoblast of the normal placenta.~• This type of tumor cell was irregular in shape but often rather cuboidal or spindle-shaped (Fig. 15). It had a large nucleus whose membrane was very irregular with many infoldings. The nucleolus was skeinlike and large. Desmosomes were observed between the trophoblastic cells (Fig. 13). The RNA was found in the form of oolvribosomes (clusters of ribosomes) . V Prv little endoplasmic reticulum could be found in this type of cell. Although the Golgi apparatus could not be found in many cells. in some cells, a large part of the cytoplasm was occupied by accumulations of Golg-i membranes and vacuoles (Fig. 10). C<·ntrisomes were occasionally found in this type of cell. The mitochondria were slightly larg-er than those of the normal trophoblastic tissue, measuring from 0.5 to 3 /t, and appearing polygonal with few cristae and abundant mitochondrial matrix (Figs. 9, 10, 12. and 13). Mitochondria of normal size and shape could be found next to the tumor n1itochondria in the same trophoblastic cell, and the mitochondria of the liver cells had a normal appearance. The cytoplasm contained variable amounts of agranular reticulum and •
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\ acunle,. '(l()H' ol \\hidt <"!lltlauwd fill(granular substanct· i Fi!_<,s. I') and H; Bundles of tonofibrils W<'re often found (Fi~-. i 2). The other type of cell was a multinucleat('(/ giant cell. Many of the~e cells wuld be regarded as syncytial masses rather than cells because of their size (Fig. 8) . The cell mt·mbrane was very irregular, and microvilli were found in many areas (Figs. 15 and 16) , especially where the tumor was invading liver sinusoids. The microvilli were short, similar to those of the syncytiotrophublast of the normal placenta (Fig. 16) . Small vacuoles at the basr· of the microvilli indicated pmocytotic activity. The nuclei resembled those of the cytotrophoblast, but they were more irregular in shape, and were packed in groups in the center of the cell (Figs. 4 and 15). The cytoplasm of this typE' of cell was occupied by numerous vacuoles or short channels on the surface of which ribosomes were attached (Fig. 16) . This type of rough surfaced endoplasmic reticulum is characteristic of the- syncytial trophoblast. However, polyribosomes free in the cytoplasm were also found in multinucleated cells. This type of cell had fewer mitochondria, was more regular in shape. and contained more cristae than those described in the first type of cell. In some of the cells the endoplasmic spaces were dilated, and in these cells the cytoplasm was rich in accumulations of fine granular ~ubstance. Lipid particles were abundant in these areas_ Tonofibrils or glycogen were not obsen:ed in the syncytial type of tumor cell. Crystals or virus particles in nuclei or cytoplasm were not observed. Most of the tumor cells belonged tc, the two described types, but some had a different
Fi11:. 9. From the iunction zone showing part of liver
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N~te the differen~e between the cytoplasm and mitochondria of these two cells. The cytopiasm of the liver (CL) contains glycogen (GL) in rosette-shaped accumulations and endophWnic reticulum (ER), while the cytoplasm of the trophoblast (CT)' contains polyri~rnes (P). The mitochondria of the trophoblast (MT) are polygonal with few aistae compared With ..the
regular mitochondria of the liver ( ML) with many .cristae. The intercellular space (IS) is narrow, and microvilli-like processes from both cells project into the space. No dcsmosomes are seen in this field. (Compare with Fig. 20.) ( x20,000.)
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Figs. 9-22. Electron micrographs.
Fig. 9. For legend see opposite page.
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ultrastructure. The cytoplasm of the !llnltinucleated type of cell often contained larg•., vacuoles or lacunae lin!:'d with microvilli (Figs. 2, 6~ and 15-.1 . rfhese intracellular spaces were also found in mononuclear troohoblastic cells. and in some cases tlw smtCf' ·-1- --- -occupied so much of the cell that the nucleus and the remaining cytoplasm were displaced to the periphery (Figs. 5 and 6). The contents of this intracellular space had a low electron density and no granular structure. The intercellular reactions in the june~ tiona! zone differed from place to place. In many areas the liver cells and tumor cells were found in close approximation without any change in the cytoplasm (Fig. 9). However, in many liver cells in contact with trophoblast, polyribosomes of tumor type were found (Fig. 11). In other places, the liwr cell cytoplasm appeared dense, and lysosomes were abundant. Proliferation of agranular reticulum was also found in liver cells at the junction zone (Fig. 20) _ A surprising observation was the presence of desmosomes between tumor and liver cells (Figs. 20 and 22). This phenomenon was found only in some parts of the junction zone, but in these areas desmosomes were numerous. Desmosome-like structures connecting neutrophil granulocytes with trophoblastic cells were also observed (Fig. 21 I . While the desmosomPs between liver and trophoblastic cells were rather characteristic. the structure found at the cell membrane of the leukocyte lacked tonofibrils., and it may be described as a thickening of the juxtaposed cell membranes rather than a desmosome. In other areas, the trophoblast exhibited phagocytic activity (Figs. 17, 18, and 19L The trophoblastic cytoplasm was filled with partly digested cellular material (Figs. 17 and 18) that vvas sometimes surrounded by a membrane (Figs. 18 and 19) which retained the characteristics of the phagocytosed celL Other parts of the trophoblastic cytoplasm contained amorphous material of medium electron density without a surrounding membrane, probably proteins from the phagocytosed cells (Fig. 17 1. .t
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In somP of tlw trophoblastic ~iar1t reils, l!d the normal pinocytotic activity of capillaries in connective tissue. In most places there was no mesenchyme between the tumor cells, but in some areas bundles of collagen with the characteristic periodicity were observed. The trophoblastic cells of the cheek pouch tumon resembled those transplanted into the liver. The former were not as well preserved, probably due to general degeneration and presence of a large amount of blood which tends to blod the fixatin'. In the cheek pouch tumors both kinds of trophoblast were present, and the cvtotrophoblastic type had the same characteristic rnitochondiia as the cells of the liver tumor. The ovaries of all tumor-bearing hamsters were enlarged with cystic folliclPs, indiCating chorionic gonadotropin production by the tumor tissue. Comment
The cytotrophoblastic type of tumor cell differed from the normal cytotrophoblastic cell not only because of the size, but also because of the very irregular mitochondria (Figs. 9, 10, 12, and 13). These changes have also been described in other malignant cells." 1 hut tlwir shapes have been explained
Fig. 10. Parts of two trophoblastic cells and a liver cell. Right: Part of cytotrophoblast with characteristic mitochondrion (J!T) and glycogen (GL). Top left: Part of liver. Between these two cells is a trophoblast which is very rich in Golgi clements (GO). (xlO,OOO.) Fig. 11. Parts of a liver cell (left) and a trophoblast (right). ML: Mitochondria of the liver cell; MB: microbody; MT: mitochondria of the trophoblast. The cytoplasm of the trophoblast is rich in po]yribosomes (long black-and-white arrow), and the same type of polyribosome is found in the cytoplasm of the liver cell (short black-and-white arrow). The cells are connected with desmosomes (D). (x35,000.)
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as artifacts because of the difficulties in fixation of malignant tissue. This cannot be the explanation here, as the other organelles of the trophoblast and the mitochondria of the liver cell in the same ultrathin sections had a normal appearance. No evidence of virus infection could bt• demonstrated, as no virus particles or intracellular crystals were found. Changes in the liver cells adjacent to the tumor. The changes found in liver cells adjacent to the tumor were few. In some places, as mentioned earlier~ proliferation of agranular reticulum was noted (Fig. 201. This phenomenon has previously been observed in liver cells exposed to toxic agents.' The functions of the agranular reticulum in liver cells seem to be associated with an enzymatic degradation of and elimination of lipid-soluble toxic agents and with the metabolism of cholesterol. The agranular reticulum mav well have manv other imnort:mt - - - - 1 - - - - - - - - fnnt·. ·---, tions not yet known. In some of the liver cells in contact with the tumor, polyribosomes of the tumor type were found. This supports the theory of Elias, Bierring, and Grunnet, 5 who suggested that transfer of RNA from tumor to normal tissue takes place. The limited areas observed and the nonspecificity of the polyribosome structure prevent drawing any definite con·· elusions. Tumor invasion compared with egg impiantation. During the study of the implantation site in the rabbit, Larsen 15 showed that a fusion of maternal and fetal cells takes p'ace at an early stage. For some hours, fetal and maternal nuclei exist in a common cytoplasm which probably also contains mitochondria "
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and other cell organelles from both imlividuals. This observation was later confirmed by Glenister, 11 who described th<:> fusion in tissue cuiture of rabbit biastocyst and endometrium. Potts~" found this stage in the implantation process of the mouse, too, but the stage lasted a very short time compared with that of the rabbit. Light microscopic studies have indicated that a similar process takes place during human implantation.''· Choriocarcinoma cells and liver cells have not been observ~ed to fuse in this way, but trophoblastic giant cells contammg liver nuclei and possibly other parts of liver cells were observed. The nuclei originating from liver cells did not show any sign of degeneration, and in this respect the~e nuclei resemble those of the uterine t'pitheliurn taken up in the trophoblast during implantation. The significance of the fusion of cytoplasmic and nuclear material during tb...e trophoblastic invasion is not known. During a discussion at the First Rochester Trophoblast Conference~" it was suggested that this process might change the immunologic characteristics of the maternal tissue and contribute to the acceptance of the trophoblast as a graft. When choriocarcinoma is implanted into hamsters, the trophoblast is rejected after 3 weeks. However, this situation is much more complicated than normal implantation, because cells from different species and from malignant tissue are involved. Desmosomes between tumor cells and the cells of the invaded tissue. The presence of desmosomes b<:>tween trophoblast and liver cells is a strang<:> phenomenon because the cells they connect are not only from different
Fig. 12. Portion of cytotrophoblast. The n1itochondria ( Ml.-.) are irregula-r in shape and have only a few cristae. The cytoplasm is rich in polyribosomes and contains vacuoles . with fine granular material (VA}. TO: Tonofibrils. (><36,000.) Fig. 13, Parts of two tumor cells. The cytoplasm contains characteristic mitochondria ( MT) and polyribosomes (P). The cells are connected with desmosomes {D). Endoplasmic reticulum is also present ( ER). (x42,000.) Fig. 14. Portion of cytotrophoblast. The cytoplasm contains Golgi apparatus (GO) and many small vesicles (VS) with medium electron-dense material. These may be part of the Golgi system or belong to the agranular reticulum. Vacuoles with many coarse granules (VA) and empty vacuoles (V E) are also found. ( <36,000.)
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Figs. 12-14. For legends see opposite page.
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organ systems, but also from difl'erent sp(~ cies--man and hamster. Desmosomes generally form between cells of same type only. An exception previously reported are the surface specializations between the oocyte and the processes from the follicular ceiL 1 The formation of desmosomes between two similar cells was regarded by Fawcett' to be a factor in selective aggregation by which separated cells in a mixed population exhibit the capacity to recognize and establish enduring contact with their own kind. Desmosomes \vere believed to be charac~ teristic for epithelial and endothelial cells, except for the intercalated discs of cardiac muscle." However, recently Ross and Greenlee2" showed attachment sites between embryonic fibroblasts in the mouse. The function of desmosomes is to keep the cells in proximity for mechanical and physiologic reasons. They play a part in organo!Tenesis in assemblin!T simrle cells into or!Tans. and they keep the cells together when external factors tend to disintegrate the tissue (trauma or edema) . Moreover, the desrnosomes may have a role as difl'usion paths for ions."" The significance of desmosomes between the trophoblastic cells and liver cells is difficult to understand. When the phenomenon is considered, it must be remembered that several difl'erent factors of importance are involved in these experiments. The invading tissue is choriocarcinoma, which has many characteristics in common with the early trophoblast. 12 This tissue is known to have the ability to interact intensively with the surrounding tissue, to invade and destroy it. Potts 2 ~ found a situation similar to ours when 0
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he studied the ult rastn1cture of mottse Jillplantation. He observt~d desmosonw-like strw·tures between the hla~tocyst and thl' uterin1· epithelium The primitive trophoblast seems to have an uniqtw ability of establishing desmosomes with difl'erent- c\'en heterologous·- cells. Thcsf· areas of contact may be the first step of the implantation in tlw normal situation and of destruction wlwn the trophoblast is malignant. Implications of phagocytosis. In some areas, v:idespread phagocytosis v:as observed (Fig. 17). This was in fields where there was considerable destruction of tissue, and many of the phag'Ocytosed cells were blood cells. This part of the tumor was more difficult to preserve, probably because of the high concentration of blood and other substances that block fixation. It is strange that phagoc.ytosis was limited only to a few fields. This may be explained by the assumption that trophoblast phagocytoses devitalized material only. The liver tissue in these areas was probably first destroyed by lack of blood supply or toxic agents from the tumor, and then secondarily ingested by the trophoblast. Phagocytic activity is not a general ability of cancer tissue, but trophoblast possesses this function. Citrastructural evidence of phagocytosis by trophoblast of human chorionic villi has not been described, but Larsen 16 observed phagocytosis by trophoblastic giant cells of the basal piate in the human piacenta. The authors are indrbted to Dr. Elliott Strauss, Miss Harriet A. McKelvey, Dr. Rarnzi S. Cotran, and Miss Christine Nin·a (Harvard Medical Sehool) for tht> Epon embedding of the liver and tumor tisstw.
Fig. 15. Portion of tumor. In the center, a tumor cell of syncytial type with large irregular nuclei (NS). In this cell, another type of nucleus is found also (NL) probably from phagocytized liver cells. The cytoplasm is dense, with large lacunae lined by microvilii. The cytotrophoblasts also have large nuclei (NT) with the characteristic trophoblm;tic skeinlike nucleoli. (x3,000.) Fig. 16. Portion of syncytial type of trophoblast. The cytoplasm contains endoplasmic reticulum
(ER), Golgi apparatus (GO) and vesicles (VE). The mitochondria (MS) are regular, but contain few cristae. The cell surface has microvilli, and small vesicles under the cell memhranc indicate pinocytotic activity (PI).
(x28~000.)
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Figs. 15-16. For legends see opposite page.
i ranspianied choriocarcinoma
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Fig. 17. Portion of trophoblast showing phagocytic activity. In the cytoplasm
(C']') is found a phagocytosed granulocyte in which the nuclrus (PG) and tnitochondria ( .~.\1G) still rnay be recognized. The amorphous substance (AS) r<>prest>nts anotlwr phagol'ytosed partly digested cell. (x18,000.)
Fig. 18. Phagocytic trophoblast. The cytoplasm (CS) contains a structure (NP) o£ the same shape and size as a liver nucleus. The granular figure NN is probably the nucleolus. The material is surrounded by lysosomes (LY). (Y8,000.)
Fig. 19. Part of two cytotrophoblastic cells from the junction zone. In the cytoplasm of the cell on the right, cytoplasmic elements (CL) from a liver cell is found (mitochondria (ML) and endoplasmic reticulum (E ..'?.). These structures are surrounded by a Inembrane ( arro\vs).
Fig. 20. Liver cell (top) and trophoblast (bottom) in contact. The liver cell is recognized by the mitochondria (ML) compared with irregular tumor mitochondria (MT). The cells are connected with desmosomes (D) and the agranular reticulum ( AR) of the liver cell is abundant near the tumor. ER: Endoplasmic reticulum; NL: nucleus of liver cell. (x20,000.)
Fig. 21. Part of neutrophil granulocyte (top) and trophoblast (bottom). The intercellular space (IS) is narrow, and there is a desmosome (D) between the cells. 1'/G: ~,Tucleus of
granulocyte: NT: nucleus of trophoblast; CT: cytoplasm of trophoblast. (x20,000.)
Fig. 22. Desmosome (D) between liver cell (top) and trophoblast (bottom). ML: Mitochondrion of liver; MT: mitochondrion of trophoblast; P: small chains of ribosomes (polyribosomes). (x40,000.)
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REFERENCES
1. Anderson, E. and ~.
3.
4. 5. 6. 7. 8. 9. 10. 11.
12.
13. 14.
15. 16.
1i.
Beams, H. W.: Ultrastructure Res. 3: 432, 1960. Bennett, H. S., and Luft, J. H.: J. Biophy3. Biochem. Cytol. 6: 113, 1959. Ehrmann, R. L., and Glisc:rman, L.: Natun• (London) 202: 404, 1964. Eckstein, P., editor: Implantation oi Ova, Mem. Soc. Endocrin. No. 6, London, 1959. Cambridge University Press. Elias, H., Bierring, F., and Grunnet, ]. : Oncologia. 18: 210, 1964. Enders, A. C.: Develop. Biol. 10: 3, 1964. Fawcett, D. W.: Expcr. C,ll. Res. Suppl. 8: 174, 1961. Fawcett, D. W.: The Cell, Philadelphia and London, 1966, W. B. Saunders Company, p. 365. Fawcett, D. W., and Selby, C. C.: J. Biophys. Biochem. Cytol. 4: 63, 1958. Ferin, J., and Gaudefroy, M., editors: Les fonctions de nidation uterine et leurs troubles, Paris, 1960, Masson et Cie. Glenister, T. W.: Nidation in Organ Culture, in The Early Conceptus, Normal and Abnormal, University of St. Andrews, London, 1965, E. & S. Livingstone, Ltd., p. 24. Hertig, A. T., and ManselL H.: The Tumors of the Female Sex Organs, Part One, Hydatidiform Mole and Choriocarcinoma, Washington, D. C., 1956, Armed Forces Institute of Pathology. Hertz, R.: Proc. Soc. Exper. Bioi. & Med. 102: 77, 1959. Knoth, 1'vf., IIesseldahl, H. and Larsen, J. F.: Ultrastructure of Human Choriocarcinoma. In preparation. Larsen,]. F.: Am. J. Anat. 109: 319, 1961. Larsen, J. F.: Electron Microscopy of the Human Placenta, in Transcript of the Second Rochester Trophoblast Conference, University of Rochester, 1963, pp. 280-300. Larsen, J. F.: Electron Microscopy of Implantation, Placentation and Fetal !'-..1enl1
18.
!9. 20.
21.
~3.
24. 25. 26. 27. on ,;o.
29. '.}().
~nJ.
branes in the Rabbit (Thesis), UnivPrsitv of Copenhagen, 1964, p. 51. Larsen, J. F.: Studies of Implantation, in Egg Implantation. CIBA Foundation Study Group No. 23, London, 1966. .J. & :\. Churchill, Ltd .. p. 82. Larsen, J. F .. and Fuchs. F.: Danish M. Bull. 10: 191, !963. Luft, J. H.: J. Biophys. Biochem. Cytol. 9: 409, 1961. Oberling. D., and Bernhard, W.: The Morphology of the Cancer Cell, in Brachet, J., and Mirsky, .\., editors: The Cell. New York and London, 1961, Academic Press, Inc .. vol. v. p. {07. Potts, M.: Implantation in Mouse and Rat, in Egg Implantation, CIBA Foundation Study Group No. 23, London, 1966, J. & A. Churchiii, Ltd., p. 89. Ross, R .. and Greenlee. T. K.: Science 153: 997, 1966. Rhodin, J. A. G., and Terzakis, J. A.: Ultrastructun· Res. 6: 88. 1962. Thiede, H. A., editor: Transcript of the First Rochester Trophoblast Conference, New York, 1961, University of Rochester. Ussing, H.: Harvey Lectures, New York and London, 1963-64, Academic Press, lric., p. l. Venable, J. H., and Coggeshalt, R.: J. Cell Bioi. 25: 407, 1965. \Vatikani, T.: 1v1IE ~f. J. 12: 43, 1962. Watson, M. L.: J. Biophys. Biochem. Cytol. 4: cJ.75, 1958, lAt .. -~
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88: 618, !964. 31. Wolstenholme, G. E. W., and O'Connor, Maeve, editors: Egg Implantation. CIBA Foundation Study Group No. 23, London, 1966. J & .'\. Churchill, Ltd. GYNEC.
245 Pond Avenue BrooklinP, Massachu.1et1, 02146
I M P L A N T A T I o N of the ovum has been studied intensively during the last few years (see reviews by Eckstein/ Ferin and Gaudefroy/0 Larsen,17 and Wolstenholme and O'Connor 31 ). These investigations have included studies of the reactions between trophoblast and invaded tissue at the ultrastructural level. 6 • 15 • 22 Electron microscopic
studies of the early human trophoblast during implantation have not yet been possible because of problems in obtaining the material. The earliest human trophoblast studied with the electron microscope is from a four-somite embryo. 19 Hertig and ManselP 2 draw attention to the resemblance between choriocarcinoma and trophoblast of the first week after implantation. Due to the lack of early human material, a study on the ultrastructure of choriocarcinoma may give information which will be useful in the understanding of trophoblastic invasion in man. Choriocarcinoma, however, is a rare tumor, and few investigators have been able to present ultrastructural observations on tumor taken directly from patients. 14 • 16 • 28 Hertz13 has been able to carry choriocarcmoma by serial passage in cheek pouches
From the Division of Electron Microscopy, University of Aarhus; The Parkway Division of The Boston Hospital for Women, Brookline; and the Department of Anatomy, University of Odense. This investigation was supported by United States Pubiic Health Service Grants No. CA 05905, HD 01261, HD 02493, and RF-44, The Lalor Foundation, The Association for the Aid of Crippled Children, and The Danish Anti-Cancer League.
1109
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I ker.mbt'r I), jYt,: \n" I Oh'>T. & ( ;y~wt.
Bierring
of hamsters, and this method made the tumor available for biologic studies. The ultrastructure of choriocarcinoma implanted into the cheek pouch was studied by vvynn and Davies/10 with special regard to endocrine implications. It is difficult to obtain well preserved tissue for electron microscopy from the cheek-pouch tumor and adjacent tissue, possibly due to the intensive destruction, or the abundance of blood which blocks fixation. Ehrmann and Gliserman 3 transplanted this tumor not only into cheek pouches but also into liver, brain, and other organs in hanlsters, and studied the junction zone with tht' light microscope. In the present investigation, the junction zone was studied with the electron microscope. Materials and methods
Experimentai series of hamsters.
~mgie
5 mm. x 2 rnm. slivers of the Green choriocarcinoma (obtained froin Dr. Roy Hertz and carried by hamster cheek pouch transfer approximately every 10 days since February, 1962) were inoculated through an abdominal incision into the livers of 26 young female 80 to 100 gram Syrian hamsters, using a Lundy-Irving caudal needle. Eighteen of these grew suitable intrahepatic turflors which provided enough tumor-liver junctional tissue for the preparation of 125 Epon bloch Although cortisone was not used for the routine cheek pouch passage of the tumor, it was necessary for the maintenance of intrahepatic tumor growth. Each experimental hamster was given an intradermal injection twice weekly, of 0.05 ml. cortisone (Cortone i (50 mg. per milliliter) . The animals \vere
Table I. Experimental series of hams ten. No. of animals
Day1 uj tumur growth
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Remarks
Lung metastases Ill I animal Lung metastases m I animal Lung metastases Ill 1 animal Lung metastases tn :l animals Lung metastases m 1 animal
------------------~---~--~----
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---------------
killed 14, 16, 17, 18, or 20 days after inoculation with tumor. The experiments are summarized in Table I. The animals were anesthetized with Nembutal and biopsies from representative areas of the junction zone ·between liver and tumor were cut down to blocks measuring approximately 1 to 2 mm.,a fixed in 1 per cent osmic acid in S-collidene buffer, 2 dehydrated in methyl alcohol and embedded in Epon.~" Sections were cut at 1 p. and stained with 1 per cent toluidine blue for light microscopy, and ultrathin sections were cut on LKB- and Porter-Blum ultramicrotomes. The sections were stained with lead citrate 27 or uranyl acetate. £!1 Electron micrographs were made with JEM-6C and Phillips-100 electron microscopes. Larger pieces of the junction zone were fixed in Bouin's solution, embedded in paraffin, and stained with hematoxylin and eosin for ordinary light In icroscopy.
Fig. 1. Junction zone parallel to the liver sinusoids. The line separating ~e liver cells (L) from trophoblast (T) is very regular. The syncytial type of cell (S) appears darker with this stain. The large tumor blood space is lined with trophoblasts. (x120.) Fig. 2. Trophoblastic cells (T) invading the liver sinU$oids. The trophoblasts contain dark corpuBcles, some of which are phagocytosed red blood cells. (x240.) Fiv_ 3. Trnnhnhla•to inv.adin.,. liver sinusoids. In some of the sinusoids the invasion takes place i~oth~- sJ>;.~;-~£-Di~~e--bt;t;~;n th~ llve~-cells (L) and the Kupffer cells (K). (x300.) Fig. 4. Portion of tumor with syncytial elements (S) and cytotrophoblast (C). Some of the blood spaces are lined with tumor tells (B), others with endothelium (V). (x120.)
Volume 99 Number 8
Transplanted choriocarcinoma
Figs. 1-8. Light mic rogra phs. Epon embedded. T oluidine blue stained.
Figs. 1-4. For legends see opposite page.
1111
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Control series of hamsters. Fifty-one yourl~.! female hamsters ( 80 to 100 ,grams) were used in four different control groups. To evaluate the changes found in the liver cells of the junction zone, it was necessary to have tissue from livers of (a) normal hamsters--12 animals; (b) hamsters treated with cortisone-13 animals; (c) hamsters with choriocarcinoma in cheek pouches-12 animals ; and (d) hamsters with choriocarcinoma in cheek pouches and treated with cortisone14 animals. The control animals had the same were anesthetized the same way, and the tissues were obtained, fixed, and embedded using the same methods. To determine whether contact with liver tissue had any specific observable effect on the implanted tumor, an electron microscopic comparison was made with cheek pouch tumor from hamsters after cortisone (control group (d) -see above) and from hamster~ which received no cortisont>.
Results The tumor ranged from 1 to 2.5 em. in diameter and was dark red m color, often with central hemorrhage and necrosis. The trophoblastic tissue was found as a 0.5 to 1 mm. spherical shell surrounding the necrosts. Light microscopy of l,u Epon-embedded sections. The tumor possessed the light microscopic features of choriocarcinoma, being composed of anastomosing cords of cytotrophoblast n1ixed with syncytiotrophoblast. Blood sinusoids lined with trophoblastic cells as well as true vessels lined with endothelium were observed. In some limited areas collagen
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was found. b11t for tbl' mo;,t iKtrt the tUJnnr tissue contained no mesencbyuw. Different types of tumor cells wt·re found. In Soine areas the trophoblast consisted of mononuclear, spindle-shaped cells (Fig~. ,.) and 6\ while other an· as were dominated by larger cells or syncytial masses (Figs. 4 and 8) . Most of the trophoblastic cells were Je,s heavily stained than the liver cells, probably because of the abundance of ribosomes and glycogen in the latter cells. However, some of the trophoblastic cells were intensively ~:tained (Fig. 4) .· and these cells were. found to be rich in endoplasmic reticulum when examined under the electron microscope (Figs. 15 and 16\. Some of the cells had a strange "foamy" cytoplasm (Figs. 6 and 7 i caused by large vacuoles, whereas other cells contained one large vacuole in tbe center of the cytoplasm displacing the nucleus (Figs. 5 and 61. The size of the tumor cells varied greatly, ranging from cytotrophoblastic cells measuring about 15 ,u to multinucleated giant cells (syncytial masses) almost 1 mm. in diameter (Fig. 8). The nuclei were very large (some up to 60 ~~ in diameter), irregular in shape, and had large conspicuous nucleoli (Figs. 2 through 81. Many mitoses were found. (Fig. 6.1 . The junction zone between the tumor and liver was found to be very straight in most sections (Fig·. 1 ) . The borderline was especially regular in areas where the direction of invasion was perpendicuiar to that of the liver sinusoids (Fig. 1). In areas where invasion took place paralle-l to the liver sinusoids, the trophoblast seemed to invade the space of Disse (Figs. 2 and 3) between the liver cells and the sinusoid cells.
Fig. 5. Portion of tumor with cytotrophoblasts (C) and lacunae lined by trophoblasts (LA). Syncytial areas (S) are also found. M: Mitosis. (x300.) Fig. 6. Portion of tumor with "foamy" cell type (F), cytotrophoblasts (C), lacunae (LA), and many mitoses (M). (x300.) Fig. 7. Portion of tumor with "foamy" cells and blood vessel (B) lined with trophoblast (T). (x420.) Fig. 8. Syncytial mass of tumor (S) with numerous large nuclei which have large nucleoli and very irregular nuclear membranes. ( x240.)
Volume 99 Number 8
Figs. 5-8. For legends see opposite page.
Transpianted choriocarcinoma 1113
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In many cases, single li\·er· cells or grou 1>S of two or three cells were found so deep in the tumor that their presence could not lw explained by tangential sectioning of an irregular borderline. These liver cells looked normal in most of the sections, but in some the cytoplasm appeared more basophilic than normal. Occasionally, degenerating fragments of probable liver cells could be seen within the tumor cytoplasm. Electron microscopy. Two different types of tumor cells were found. The most common type resembled the cytotrophoblast of the normal placenta.~• This type of tumor cell was irregular in shape but often rather cuboidal or spindle-shaped (Fig. 15). It had a large nucleus whose membrane was very irregular with many infoldings. The nucleolus was skeinlike and large. Desmosomes were observed between the trophoblastic cells (Fig. 13). The RNA was found in the form of oolvribosomes (clusters of ribosomes) . V Prv little endoplasmic reticulum could be found in this type of cell. Although the Golgi apparatus could not be found in many cells. in some cells, a large part of the cytoplasm was occupied by accumulations of Golg-i membranes and vacuoles (Fig. 10). C<·ntrisomes were occasionally found in this type of cell. The mitochondria were slightly larg-er than those of the normal trophoblastic tissue, measuring from 0.5 to 3 /t, and appearing polygonal with few cristae and abundant mitochondrial matrix (Figs. 9, 10, 12. and 13). Mitochondria of normal size and shape could be found next to the tumor n1itochondria in the same trophoblastic cell, and the mitochondria of the liver cells had a normal appearance. The cytoplasm contained variable amounts of agranular reticulum and •
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\ acunle,. '(l()H' ol \\hidt <"!lltlauwd fill(granular substanct· i Fi!_<,s. I') and H; Bundles of tonofibrils W<'re often found (Fi~-. i 2). The other type of cell was a multinucleat('(/ giant cell. Many of the~e cells wuld be regarded as syncytial masses rather than cells because of their size (Fig. 8) . The cell mt·mbrane was very irregular, and microvilli were found in many areas (Figs. 15 and 16) , especially where the tumor was invading liver sinusoids. The microvilli were short, similar to those of the syncytiotrophublast of the normal placenta (Fig. 16) . Small vacuoles at the basr· of the microvilli indicated pmocytotic activity. The nuclei resembled those of the cytotrophoblast, but they were more irregular in shape, and were packed in groups in the center of the cell (Figs. 4 and 15). The cytoplasm of this typE' of cell was occupied by numerous vacuoles or short channels on the surface of which ribosomes were attached (Fig. 16) . This type of rough surfaced endoplasmic reticulum is characteristic of the- syncytial trophoblast. However, polyribosomes free in the cytoplasm were also found in multinucleated cells. This type of cell had fewer mitochondria, was more regular in shape. and contained more cristae than those described in the first type of cell. In some of the cells the endoplasmic spaces were dilated, and in these cells the cytoplasm was rich in accumulations of fine granular ~ubstance. Lipid particles were abundant in these areas_ Tonofibrils or glycogen were not obsen:ed in the syncytial type of tumor cell. Crystals or virus particles in nuclei or cytoplasm were not observed. Most of the tumor cells belonged tc, the two described types, but some had a different
Fi11:. 9. From the iunction zone showing part of liver
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(top) a(!d cytotrophoblast (bottom).
N~te the differen~e between the cytoplasm and mitochondria of these two cells. The cytopiasm of the liver (CL) contains glycogen (GL) in rosette-shaped accumulations and endophWnic reticulum (ER), while the cytoplasm of the trophoblast (CT)' contains polyri~rnes (P). The mitochondria of the trophoblast (MT) are polygonal with few aistae compared With ..the
regular mitochondria of the liver ( ML) with many .cristae. The intercellular space (IS) is narrow, and microvilli-like processes from both cells project into the space. No dcsmosomes are seen in this field. (Compare with Fig. 20.) ( x20,000.)
Volume 99 Number 8
Figs. 9-22. Electron micrographs.
Fig. 9. For legend see opposite page.
Transplanted choriocarcinoma 1115
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ultrastructure. The cytoplasm of the !llnltinucleated type of cell often contained larg•., vacuoles or lacunae lin!:'d with microvilli (Figs. 2, 6~ and 15-.1 . rfhese intracellular spaces were also found in mononuclear troohoblastic cells. and in some cases tlw smtCf' ·-1- --- -occupied so much of the cell that the nucleus and the remaining cytoplasm were displaced to the periphery (Figs. 5 and 6). The contents of this intracellular space had a low electron density and no granular structure. The intercellular reactions in the june~ tiona! zone differed from place to place. In many areas the liver cells and tumor cells were found in close approximation without any change in the cytoplasm (Fig. 9). However, in many liver cells in contact with trophoblast, polyribosomes of tumor type were found (Fig. 11). In other places, the liwr cell cytoplasm appeared dense, and lysosomes were abundant. Proliferation of agranular reticulum was also found in liver cells at the junction zone (Fig. 20) _ A surprising observation was the presence of desmosomes between tumor and liver cells (Figs. 20 and 22). This phenomenon was found only in some parts of the junction zone, but in these areas desmosomes were numerous. Desmosome-like structures connecting neutrophil granulocytes with trophoblastic cells were also observed (Fig. 21 I . While the desmosomPs between liver and trophoblastic cells were rather characteristic. the structure found at the cell membrane of the leukocyte lacked tonofibrils., and it may be described as a thickening of the juxtaposed cell membranes rather than a desmosome. In other areas, the trophoblast exhibited phagocytic activity (Figs. 17, 18, and 19L The trophoblastic cytoplasm was filled with partly digested cellular material (Figs. 17 and 18) that vvas sometimes surrounded by a membrane (Figs. 18 and 19) which retained the characteristics of the phagocytosed celL Other parts of the trophoblastic cytoplasm contained amorphous material of medium electron density without a surrounding membrane, probably proteins from the phagocytosed cells (Fig. 17 1. .t
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The cytotrophoblastic type of tumor cell differed from the normal cytotrophoblastic cell not only because of the size, but also because of the very irregular mitochondria (Figs. 9, 10, 12, and 13). These changes have also been described in other malignant cells." 1 hut tlwir shapes have been explained
Fig. 10. Parts of two trophoblastic cells and a liver cell. Right: Part of cytotrophoblast with characteristic mitochondrion (J!T) and glycogen (GL). Top left: Part of liver. Between these two cells is a trophoblast which is very rich in Golgi clements (GO). (xlO,OOO.) Fig. 11. Parts of a liver cell (left) and a trophoblast (right). ML: Mitochondria of the liver cell; MB: microbody; MT: mitochondria of the trophoblast. The cytoplasm of the trophoblast is rich in po]yribosomes (long black-and-white arrow), and the same type of polyribosome is found in the cytoplasm of the liver cell (short black-and-white arrow). The cells are connected with desmosomes (D). (x35,000.)
1118
Larsen, Ehrmannn, and
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as artifacts because of the difficulties in fixation of malignant tissue. This cannot be the explanation here, as the other organelles of the trophoblast and the mitochondria of the liver cell in the same ultrathin sections had a normal appearance. No evidence of virus infection could bt• demonstrated, as no virus particles or intracellular crystals were found. Changes in the liver cells adjacent to the tumor. The changes found in liver cells adjacent to the tumor were few. In some places, as mentioned earlier~ proliferation of agranular reticulum was noted (Fig. 201. This phenomenon has previously been observed in liver cells exposed to toxic agents.' The functions of the agranular reticulum in liver cells seem to be associated with an enzymatic degradation of and elimination of lipid-soluble toxic agents and with the metabolism of cholesterol. The agranular reticulum mav well have manv other imnort:mt - - - - 1 - - - - - - - - fnnt·. ·---, tions not yet known. In some of the liver cells in contact with the tumor, polyribosomes of the tumor type were found. This supports the theory of Elias, Bierring, and Grunnet, 5 who suggested that transfer of RNA from tumor to normal tissue takes place. The limited areas observed and the nonspecificity of the polyribosome structure prevent drawing any definite con·· elusions. Tumor invasion compared with egg impiantation. During the study of the implantation site in the rabbit, Larsen 15 showed that a fusion of maternal and fetal cells takes p'ace at an early stage. For some hours, fetal and maternal nuclei exist in a common cytoplasm which probably also contains mitochondria "
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and other cell organelles from both imlividuals. This observation was later confirmed by Glenister, 11 who described th<:> fusion in tissue cuiture of rabbit biastocyst and endometrium. Potts~" found this stage in the implantation process of the mouse, too, but the stage lasted a very short time compared with that of the rabbit. Light microscopic studies have indicated that a similar process takes place during human implantation.''· Choriocarcinoma cells and liver cells have not been observ~ed to fuse in this way, but trophoblastic giant cells contammg liver nuclei and possibly other parts of liver cells were observed. The nuclei originating from liver cells did not show any sign of degeneration, and in this respect the~e nuclei resemble those of the uterine t'pitheliurn taken up in the trophoblast during implantation. The significance of the fusion of cytoplasmic and nuclear material during tb...e trophoblastic invasion is not known. During a discussion at the First Rochester Trophoblast Conference~" it was suggested that this process might change the immunologic characteristics of the maternal tissue and contribute to the acceptance of the trophoblast as a graft. When choriocarcinoma is implanted into hamsters, the trophoblast is rejected after 3 weeks. However, this situation is much more complicated than normal implantation, because cells from different species and from malignant tissue are involved. Desmosomes between tumor cells and the cells of the invaded tissue. The presence of desmosomes b<:>tween trophoblast and liver cells is a strang<:> phenomenon because the cells they connect are not only from different
Fig. 12. Portion of cytotrophoblast. The n1itochondria ( Ml.-.) are irregula-r in shape and have only a few cristae. The cytoplasm is rich in polyribosomes and contains vacuoles . with fine granular material (VA}. TO: Tonofibrils. (><36,000.) Fig. 13, Parts of two tumor cells. The cytoplasm contains characteristic mitochondria ( MT) and polyribosomes (P). The cells are connected with desmosomes {D). Endoplasmic reticulum is also present ( ER). (x42,000.) Fig. 14. Portion of cytotrophoblast. The cytoplasm contains Golgi apparatus (GO) and many small vesicles (VS) with medium electron-dense material. These may be part of the Golgi system or belong to the agranular reticulum. Vacuoles with many coarse granules (VA) and empty vacuoles (V E) are also found. ( <36,000.)
Voiume 99 Number 8
Figs. 12-14. For legends see opposite page.
Transplanied choriocarcinoma 1119
i i 20
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Biemng ''ll
organ systems, but also from difl'erent sp(~ cies--man and hamster. Desmosomes generally form between cells of same type only. An exception previously reported are the surface specializations between the oocyte and the processes from the follicular ceiL 1 The formation of desmosomes between two similar cells was regarded by Fawcett' to be a factor in selective aggregation by which separated cells in a mixed population exhibit the capacity to recognize and establish enduring contact with their own kind. Desmosomes \vere believed to be charac~ teristic for epithelial and endothelial cells, except for the intercalated discs of cardiac muscle." However, recently Ross and Greenlee2" showed attachment sites between embryonic fibroblasts in the mouse. The function of desmosomes is to keep the cells in proximity for mechanical and physiologic reasons. They play a part in organo!Tenesis in assemblin!T simrle cells into or!Tans. and they keep the cells together when external factors tend to disintegrate the tissue (trauma or edema) . Moreover, the desrnosomes may have a role as difl'usion paths for ions."" The significance of desmosomes between the trophoblastic cells and liver cells is difficult to understand. When the phenomenon is considered, it must be remembered that several difl'erent factors of importance are involved in these experiments. The invading tissue is choriocarcinoma, which has many characteristics in common with the early trophoblast. 12 This tissue is known to have the ability to interact intensively with the surrounding tissue, to invade and destroy it. Potts 2 ~ found a situation similar to ours when 0
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he studied the ult rastn1cture of mottse Jillplantation. He observt~d desmosonw-like strw·tures between the hla~tocyst and thl' uterin1· epithelium The primitive trophoblast seems to have an uniqtw ability of establishing desmosomes with difl'erent- c\'en heterologous·- cells. Thcsf· areas of contact may be the first step of the implantation in tlw normal situation and of destruction wlwn the trophoblast is malignant. Implications of phagocytosis. In some areas, v:idespread phagocytosis v:as observed (Fig. 17). This was in fields where there was considerable destruction of tissue, and many of the phag'Ocytosed cells were blood cells. This part of the tumor was more difficult to preserve, probably because of the high concentration of blood and other substances that block fixation. It is strange that phagoc.ytosis was limited only to a few fields. This may be explained by the assumption that trophoblast phagocytoses devitalized material only. The liver tissue in these areas was probably first destroyed by lack of blood supply or toxic agents from the tumor, and then secondarily ingested by the trophoblast. Phagocytic activity is not a general ability of cancer tissue, but trophoblast possesses this function. Citrastructural evidence of phagocytosis by trophoblast of human chorionic villi has not been described, but Larsen 16 observed phagocytosis by trophoblastic giant cells of the basal piate in the human piacenta. The authors are indrbted to Dr. Elliott Strauss, Miss Harriet A. McKelvey, Dr. Rarnzi S. Cotran, and Miss Christine Nin·a (Harvard Medical Sehool) for tht> Epon embedding of the liver and tumor tisstw.
Fig. 15. Portion of tumor. In the center, a tumor cell of syncytial type with large irregular nuclei (NS). In this cell, another type of nucleus is found also (NL) probably from phagocytized liver cells. The cytoplasm is dense, with large lacunae lined by microvilii. The cytotrophoblasts also have large nuclei (NT) with the characteristic trophoblm;tic skeinlike nucleoli. (x3,000.) Fig. 16. Portion of syncytial type of trophoblast. The cytoplasm contains endoplasmic reticulum
(ER), Golgi apparatus (GO) and vesicles (VE). The mitochondria (MS) are regular, but contain few cristae. The cell surface has microvilli, and small vesicles under the cell memhranc indicate pinocytotic activity (PI).
(x28~000.)
Voiume 99 :\lumber 8
Figs. 15-16. For legends see opposite page.
i ranspianied choriocarcinoma
,,"'. ll.l
22
larsen, Ehrmannn, and
Bierring
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Ob'it. & Gynt-•·.
Fig. 17. Portion of trophoblast showing phagocytic activity. In the cytoplasm
(C']') is found a phagocytosed granulocyte in which the nuclrus (PG) and tnitochondria ( .~.\1G) still rnay be recognized. The amorphous substance (AS) r<>prest>nts anotlwr phagol'ytosed partly digested cell. (x18,000.)
Fig. 18. Phagocytic trophoblast. The cytoplasm (CS) contains a structure (NP) o£ the same shape and size as a liver nucleus. The granular figure NN is probably the nucleolus. The material is surrounded by lysosomes (LY). (Y8,000.)
Fig. 19. Part of two cytotrophoblastic cells from the junction zone. In the cytoplasm of the cell on the right, cytoplasmic elements (CL) from a liver cell is found (mitochondria (ML) and endoplasmic reticulum (E ..'?.). These structures are surrounded by a Inembrane ( arro\vs).
Fig. 20. Liver cell (top) and trophoblast (bottom) in contact. The liver cell is recognized by the mitochondria (ML) compared with irregular tumor mitochondria (MT). The cells are connected with desmosomes (D) and the agranular reticulum ( AR) of the liver cell is abundant near the tumor. ER: Endoplasmic reticulum; NL: nucleus of liver cell. (x20,000.)
Fig. 21. Part of neutrophil granulocyte (top) and trophoblast (bottom). The intercellular space (IS) is narrow, and there is a desmosome (D) between the cells. 1'/G: ~,Tucleus of
granulocyte: NT: nucleus of trophoblast; CT: cytoplasm of trophoblast. (x20,000.)
Fig. 22. Desmosome (D) between liver cell (top) and trophoblast (bottom). ML: Mitochondrion of liver; MT: mitochondrion of trophoblast; P: small chains of ribosomes (polyribosomes). (x40,000.)
1124
Larsen, Ehrmannn, and
Bierring
REFERENCES
1. Anderson, E. and ~.
3.
4. 5. 6. 7. 8. 9. 10. 11.
12.
13. 14.
15. 16.
1i.
Beams, H. W.: Ultrastructure Res. 3: 432, 1960. Bennett, H. S., and Luft, J. H.: J. Biophy3. Biochem. Cytol. 6: 113, 1959. Ehrmann, R. L., and Glisc:rman, L.: Natun• (London) 202: 404, 1964. Eckstein, P., editor: Implantation oi Ova, Mem. Soc. Endocrin. No. 6, London, 1959. Cambridge University Press. Elias, H., Bierring, F., and Grunnet, ]. : Oncologia. 18: 210, 1964. Enders, A. C.: Develop. Biol. 10: 3, 1964. Fawcett, D. W.: Expcr. C,ll. Res. Suppl. 8: 174, 1961. Fawcett, D. W.: The Cell, Philadelphia and London, 1966, W. B. Saunders Company, p. 365. Fawcett, D. W., and Selby, C. C.: J. Biophys. Biochem. Cytol. 4: 63, 1958. Ferin, J., and Gaudefroy, M., editors: Les fonctions de nidation uterine et leurs troubles, Paris, 1960, Masson et Cie. Glenister, T. W.: Nidation in Organ Culture, in The Early Conceptus, Normal and Abnormal, University of St. Andrews, London, 1965, E. & S. Livingstone, Ltd., p. 24. Hertig, A. T., and ManselL H.: The Tumors of the Female Sex Organs, Part One, Hydatidiform Mole and Choriocarcinoma, Washington, D. C., 1956, Armed Forces Institute of Pathology. Hertz, R.: Proc. Soc. Exper. Bioi. & Med. 102: 77, 1959. Knoth, 1'vf., IIesseldahl, H. and Larsen, J. F.: Ultrastructure of Human Choriocarcinoma. In preparation. Larsen,]. F.: Am. J. Anat. 109: 319, 1961. Larsen, J. F.: Electron Microscopy of the Human Placenta, in Transcript of the Second Rochester Trophoblast Conference, University of Rochester, 1963, pp. 280-300. Larsen, J. F.: Electron Microscopy of Implantation, Placentation and Fetal !'-..1enl1
18.
!9. 20.
21.
~3.
24. 25. 26. 27. on ,;o.
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branes in the Rabbit (Thesis), UnivPrsitv of Copenhagen, 1964, p. 51. Larsen, J. F.: Studies of Implantation, in Egg Implantation. CIBA Foundation Study Group No. 23, London, 1966. .J. & :\. Churchill, Ltd .. p. 82. Larsen, J. F .. and Fuchs. F.: Danish M. Bull. 10: 191, !963. Luft, J. H.: J. Biophys. Biochem. Cytol. 9: 409, 1961. Oberling. D., and Bernhard, W.: The Morphology of the Cancer Cell, in Brachet, J., and Mirsky, .\., editors: The Cell. New York and London, 1961, Academic Press, Inc .. vol. v. p. {07. Potts, M.: Implantation in Mouse and Rat, in Egg Implantation, CIBA Foundation Study Group No. 23, London, 1966, J. & A. Churchiii, Ltd., p. 89. Ross, R .. and Greenlee. T. K.: Science 153: 997, 1966. Rhodin, J. A. G., and Terzakis, J. A.: Ultrastructun· Res. 6: 88. 1962. Thiede, H. A., editor: Transcript of the First Rochester Trophoblast Conference, New York, 1961, University of Rochester. Ussing, H.: Harvey Lectures, New York and London, 1963-64, Academic Press, lric., p. l. Venable, J. H., and Coggeshalt, R.: J. Cell Bioi. 25: 407, 1965. \Vatikani, T.: 1v1IE ~f. J. 12: 43, 1962. Watson, M. L.: J. Biophys. Biochem. Cytol. 4: cJ.75, 1958, lAt .. -~
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