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Intercellular junctions between Novikoff hepatoma cells and hepatic cells
Copyright
A/l rights
Q 1972 by Academic Press, Inc. of reproduction in any form reserwd
Experimental Cell Research 74 (1972) 355-358
INTERCELLULAR
JUNCTIONS BETWEEN NOVIKOFF AND HEPATIC CELLS G. TREMBLAY’
Dipartement
de Pathologie,
Facultk de Midecine,
HEPATOMA
CELLS
and F. BABAI Universitk
de Montrkal,
Montrdal
101, Canada
SUMMARY In the course of an ultrastructural study of liver invasion by Novikoff hepatoma transplanted in rat liver, several instances of intercellular junctions between tumor cells and non-neoplastic hepatic cells were noted. Such junctions were of the macula adherens type. This finding is interpreted as evidence of differentiation of the tumor cells.
Adjacent normal epithelial cells maintain cells and non-neoplastic hepatic cells were extensive contact through various types of noted. This observation of junctions between specialized intercellular junctions. There is a transplanted tumor and host cells is the increasing evidence that, in malignant epi- object of the present report. thelial tumors, the number of junctional elements is reduced, although the extent of MATERIALS AND METHODS the decreaseappears to vary for the different types of junctions. Thus, an absence of tight Male Sprague-Dawley rats weighing from 180 to g were used. Celiotomy was performed under junctions and a scarcity of nexuses (gap 250 ether anesthesia and a small fragment (1 mm) of junctions) has been demonstrated in some solid Novikoff hepatoma was implanted in the left lobe of the liver as described previously [I]. Seven carcinomas [7, 81.On the other hand, desmo- to 10 days later, the animals were perfused via the somes and intermediate junctions between thoracic aorta for 1 to 2 min with Ringer solution containing 0.1 % procain and 5 mg/lOO ml of heparin tumor cells have been observed in a variety [4] and for 15 min with 2.5 9b glutaraldehyde in of malignant neoplasms, although their num- phosphate buffer, pH 7.4 [5]. After perfusion, small containing tumor tissue and surrounding ber appears to be less than in normal cor- fragments liver parenchyma were immersed for 2 to 3 h in the responding tissues [7]. In contrast to this, same fixative, washed in phosphate buffer containing 0.25 M sucrose, and postfixed for 1 h in 1 “b osmium the development of junctions between malig- tetroxide in phosphate buffer. The tissues were nant cells and non-neoplastic cells has never dehydrated in graded concentrations of alcohols and embedded in Araldite. Thin sections were cut been conclusively demonstrated. with glass knives on a Porter-Blum MT I ultramicroIn the course of a study on invasion of tome, stained with uranyl acetate and lead citrate, liver by Novikoff hepatoma, several instances and examined with a Philips 200 electron microscope. of intercellular junctions between tumor RESULTS I Present address: Department of Pathology, Faculty of Medicine, McGill University, Montreal 110, Canada.
As reported elsewhere [2], the tumor cells in the zone of invasion often had irregular Exptl
Cell lies 74 (1972)
356
G. Tremblay & F. Babai
contours with formation of cytoplasmic processesextending into adjacent hepatic cells. The interface between tumor cells and adjacent hepatic cells was of diverse aspects. The plasma membranes of the two types of cells in most areas remained separated by a gap of varying width while, in other regions, the membranes were in close proximity. Moreover, in several instances, tumor cells and adjacent hepatic cells were found to be attached by specialized junctional elements. These intercellular junctions were of the macula adherens type characterized by parallel and straight arrangement of the opposing cell membranes, increased electron-opaque material in the intercellular space, and condensation of the subjacent cytoplasm both in the hepatic and in the tumor cells (figs 1, 2). Some of the junctions were imperfectly defined but most were well-developed structures (fig. 2). The distribution of the junctions in relation to the perimeter of the tumor cells showed no regular pattern, but groups of several maculae adhaerentes sometimes appeared concentrated in one area (fig. 1). In the hepatic cells, the junctions seemedto be chiefly located close to or at least in the vicinity of bile canaliculi (figs 1, 2). Adjacent tumor cells usually remained separated by a clear intercellular space but, occasionally, they were joined by maculae adhaerentes (fig. 3). Furthermore, junctional elements were observed between tumor cells in which the intercellular space was obliterated, although it could not be determined whether these junctions were of the tight or nexus type (fig. 3).
DISCUSSION This study demonstrates unequivocally the development of junctions between malignant tumor cells and non-neoplastic cells. In a recent ultrastructural study of metastatic growth by Yoshida hepatoma in chick embryo liver, Locker et al. [6] observed that hepatic cells and tumor cells in intimate contact occasionally showed a thickening of their respective opposing cell membrane. These authors suggested the possibility that this thickening might represent a kind of rudimentary junctional complex. In the present observation, most of the junctions between tumor cells and hepatic cells were well developed and had a characteristic appearance, leaving no doubt as to their nature. In Paget’s disease of the breast, desmosomes joining tumor cells and adjacent keratinizing epidermal cells were observed by Sagebiel [lo]. However, as was pointed out, the origin of the Paget cells remains controversial and it could not be ascertained whether the desmosomesrepresented residual junctions of transformed epidermal cells or desmosomes formed after migration or metastasis [lo]. In the present study, the fact that the junctions occurred between liver cells and transplanted tumor cells demonstrates that they are not residual junctions but newly formed structures. While the mechanism of junction genesisis still obscure, it is generally accepted that these cell attachments play a role in forming and maintaining specific characteristics of
Fig. I. Electron micrograph showing a tumor cell (T) attached to two hepatic cells (HI, I%*) by four junctions
(arrows). A macula adherens (MA) also connects the two hepatic cells near a bile canaliculus (BC). x 11 000. Fig. 2. Higher magnification of the interface between the tumor cell (T) and the two hepatic cells (H,, Hz).
Two maculae adhaerentes show the characteristic condensation of the subjacent cytoplasm, both in the tumor and in the hepatic cells. x 26 000. Fig. 3. Two tumor cells (T) are linked by two maculae adhaerentes (arrows). Between these two junctions, there is also a junctional element, with obliteration of the intercellular space. x 25 000. Exptl Cell Res 74 (1972)
Junctions between Nocikojf hepatoma and hepatic cells
357
E.rptl Cell RES.74 c/97.?)
358
G. Tremblay & F. Babai
tissues [3]. The formation of cell attachments with non-neoplastic cells can be considered as an indication of differentiation of the tumor cells and thus adds support to the concept that some of the progeny of malignant cells have the capability of differentiation [9]. Indeed, as pointed out recently by Pierce & Wallace [9], there is growing evidence that the variable histological appearance of tumors is attributable to relative degrees of differentiation rather than to relative degrees of dedifferentiation. One interesting point in the present study is that the formation of cell junctions necessitated collaboration and interaction of some kind between neoplastic and normal cells. The exact nature of this interaction remains unknown. It is possible that spontaneous differentiation of the tumor cells leads to their recognition and binding by the adjacent hepatic cells. However, it must be pointed out that the cells with junctions were otherwise similar in ultrastructural appearance to the cells without attachment, so that the morphological manifestation of differentiation would have been limited to the cell surface. Another, though speculative, possibility is that the hepatic cells played a role in the
Exptl
Cell Res 74 (1972)
induction of differentiation evolving in the formation of junctions by the tumor cells. More information is needed concerning normal and abnormal cell-to-cell interactions before these questions can be answered. The excellent technical assistance of Miss Francoise Peloquin is gratefully acknowledged. This study was supported by a grant from the National Cancer Institute of Canada.
REFERENCES 1. Babai, F, Tremblay, G & Dumont, A, J ultrastruct res 28 (1969) 125. 2. Babai, F & Tremblay, G. Submitted for publication. 3. Campbell, R D & Campbell, J H, Results and problems in cell differentiation (ed J Reinert & H Ursprung) vol. 2, p. 261. Springer-Verlag, Berlin (1971). 4. Forssmann, W G, Siegrist, G, Orci, L, Girardier, L, Pictet, R & Rouiller, C, J microscopic 6 (1967) 279. 5. Karlsson, U & Schultz, R L, J ultrastruct res 12 (1965) 160. 6. Locker, J, Goldblatt, P J & Leighton, J, Cancer res 30 (1970) 1632. 7. Martinez-Palomo, A, Lab invest 22 (1970) 605. 8. McNutt, N S & Weinstein, R S, Science 165 (1969) 597. 9. Pierce, G B & Wallace, C, Cancer res 31 (1971)
127. 10. Sagebiel, R W, Am j path 57 (1969) 49. Received February 15, 1972
A/l rights
Q 1972 by Academic Press, Inc. of reproduction in any form reserwd
Experimental Cell Research 74 (1972) 355-358
INTERCELLULAR
JUNCTIONS BETWEEN NOVIKOFF AND HEPATIC CELLS G. TREMBLAY’
Dipartement
de Pathologie,
Facultk de Midecine,
HEPATOMA
CELLS
and F. BABAI Universitk
de Montrkal,
Montrdal
101, Canada
SUMMARY In the course of an ultrastructural study of liver invasion by Novikoff hepatoma transplanted in rat liver, several instances of intercellular junctions between tumor cells and non-neoplastic hepatic cells were noted. Such junctions were of the macula adherens type. This finding is interpreted as evidence of differentiation of the tumor cells.
Adjacent normal epithelial cells maintain cells and non-neoplastic hepatic cells were extensive contact through various types of noted. This observation of junctions between specialized intercellular junctions. There is a transplanted tumor and host cells is the increasing evidence that, in malignant epi- object of the present report. thelial tumors, the number of junctional elements is reduced, although the extent of MATERIALS AND METHODS the decreaseappears to vary for the different types of junctions. Thus, an absence of tight Male Sprague-Dawley rats weighing from 180 to g were used. Celiotomy was performed under junctions and a scarcity of nexuses (gap 250 ether anesthesia and a small fragment (1 mm) of junctions) has been demonstrated in some solid Novikoff hepatoma was implanted in the left lobe of the liver as described previously [I]. Seven carcinomas [7, 81.On the other hand, desmo- to 10 days later, the animals were perfused via the somes and intermediate junctions between thoracic aorta for 1 to 2 min with Ringer solution containing 0.1 % procain and 5 mg/lOO ml of heparin tumor cells have been observed in a variety [4] and for 15 min with 2.5 9b glutaraldehyde in of malignant neoplasms, although their num- phosphate buffer, pH 7.4 [5]. After perfusion, small containing tumor tissue and surrounding ber appears to be less than in normal cor- fragments liver parenchyma were immersed for 2 to 3 h in the responding tissues [7]. In contrast to this, same fixative, washed in phosphate buffer containing 0.25 M sucrose, and postfixed for 1 h in 1 “b osmium the development of junctions between malig- tetroxide in phosphate buffer. The tissues were nant cells and non-neoplastic cells has never dehydrated in graded concentrations of alcohols and embedded in Araldite. Thin sections were cut been conclusively demonstrated. with glass knives on a Porter-Blum MT I ultramicroIn the course of a study on invasion of tome, stained with uranyl acetate and lead citrate, liver by Novikoff hepatoma, several instances and examined with a Philips 200 electron microscope. of intercellular junctions between tumor RESULTS I Present address: Department of Pathology, Faculty of Medicine, McGill University, Montreal 110, Canada.
As reported elsewhere [2], the tumor cells in the zone of invasion often had irregular Exptl
Cell lies 74 (1972)
356
G. Tremblay & F. Babai
contours with formation of cytoplasmic processesextending into adjacent hepatic cells. The interface between tumor cells and adjacent hepatic cells was of diverse aspects. The plasma membranes of the two types of cells in most areas remained separated by a gap of varying width while, in other regions, the membranes were in close proximity. Moreover, in several instances, tumor cells and adjacent hepatic cells were found to be attached by specialized junctional elements. These intercellular junctions were of the macula adherens type characterized by parallel and straight arrangement of the opposing cell membranes, increased electron-opaque material in the intercellular space, and condensation of the subjacent cytoplasm both in the hepatic and in the tumor cells (figs 1, 2). Some of the junctions were imperfectly defined but most were well-developed structures (fig. 2). The distribution of the junctions in relation to the perimeter of the tumor cells showed no regular pattern, but groups of several maculae adhaerentes sometimes appeared concentrated in one area (fig. 1). In the hepatic cells, the junctions seemedto be chiefly located close to or at least in the vicinity of bile canaliculi (figs 1, 2). Adjacent tumor cells usually remained separated by a clear intercellular space but, occasionally, they were joined by maculae adhaerentes (fig. 3). Furthermore, junctional elements were observed between tumor cells in which the intercellular space was obliterated, although it could not be determined whether these junctions were of the tight or nexus type (fig. 3).
DISCUSSION This study demonstrates unequivocally the development of junctions between malignant tumor cells and non-neoplastic cells. In a recent ultrastructural study of metastatic growth by Yoshida hepatoma in chick embryo liver, Locker et al. [6] observed that hepatic cells and tumor cells in intimate contact occasionally showed a thickening of their respective opposing cell membrane. These authors suggested the possibility that this thickening might represent a kind of rudimentary junctional complex. In the present observation, most of the junctions between tumor cells and hepatic cells were well developed and had a characteristic appearance, leaving no doubt as to their nature. In Paget’s disease of the breast, desmosomes joining tumor cells and adjacent keratinizing epidermal cells were observed by Sagebiel [lo]. However, as was pointed out, the origin of the Paget cells remains controversial and it could not be ascertained whether the desmosomesrepresented residual junctions of transformed epidermal cells or desmosomes formed after migration or metastasis [lo]. In the present study, the fact that the junctions occurred between liver cells and transplanted tumor cells demonstrates that they are not residual junctions but newly formed structures. While the mechanism of junction genesisis still obscure, it is generally accepted that these cell attachments play a role in forming and maintaining specific characteristics of
Fig. I. Electron micrograph showing a tumor cell (T) attached to two hepatic cells (HI, I%*) by four junctions
(arrows). A macula adherens (MA) also connects the two hepatic cells near a bile canaliculus (BC). x 11 000. Fig. 2. Higher magnification of the interface between the tumor cell (T) and the two hepatic cells (H,, Hz).
Two maculae adhaerentes show the characteristic condensation of the subjacent cytoplasm, both in the tumor and in the hepatic cells. x 26 000. Fig. 3. Two tumor cells (T) are linked by two maculae adhaerentes (arrows). Between these two junctions, there is also a junctional element, with obliteration of the intercellular space. x 25 000. Exptl Cell Res 74 (1972)
Junctions between Nocikojf hepatoma and hepatic cells
357
E.rptl Cell RES.74 c/97.?)
358
G. Tremblay & F. Babai
tissues [3]. The formation of cell attachments with non-neoplastic cells can be considered as an indication of differentiation of the tumor cells and thus adds support to the concept that some of the progeny of malignant cells have the capability of differentiation [9]. Indeed, as pointed out recently by Pierce & Wallace [9], there is growing evidence that the variable histological appearance of tumors is attributable to relative degrees of differentiation rather than to relative degrees of dedifferentiation. One interesting point in the present study is that the formation of cell junctions necessitated collaboration and interaction of some kind between neoplastic and normal cells. The exact nature of this interaction remains unknown. It is possible that spontaneous differentiation of the tumor cells leads to their recognition and binding by the adjacent hepatic cells. However, it must be pointed out that the cells with junctions were otherwise similar in ultrastructural appearance to the cells without attachment, so that the morphological manifestation of differentiation would have been limited to the cell surface. Another, though speculative, possibility is that the hepatic cells played a role in the
Exptl
Cell Res 74 (1972)
induction of differentiation evolving in the formation of junctions by the tumor cells. More information is needed concerning normal and abnormal cell-to-cell interactions before these questions can be answered. The excellent technical assistance of Miss Francoise Peloquin is gratefully acknowledged. This study was supported by a grant from the National Cancer Institute of Canada.
REFERENCES 1. Babai, F, Tremblay, G & Dumont, A, J ultrastruct res 28 (1969) 125. 2. Babai, F & Tremblay, G. Submitted for publication. 3. Campbell, R D & Campbell, J H, Results and problems in cell differentiation (ed J Reinert & H Ursprung) vol. 2, p. 261. Springer-Verlag, Berlin (1971). 4. Forssmann, W G, Siegrist, G, Orci, L, Girardier, L, Pictet, R & Rouiller, C, J microscopic 6 (1967) 279. 5. Karlsson, U & Schultz, R L, J ultrastruct res 12 (1965) 160. 6. Locker, J, Goldblatt, P J & Leighton, J, Cancer res 30 (1970) 1632. 7. Martinez-Palomo, A, Lab invest 22 (1970) 605. 8. McNutt, N S & Weinstein, R S, Science 165 (1969) 597. 9. Pierce, G B & Wallace, C, Cancer res 31 (1971)
127. 10. Sagebiel, R W, Am j path 57 (1969) 49. Received February 15, 1972