13

The Characteristics of Cell Lines Derived from Human Germ Cell

Tumors

Peter W. Andrews

Introduction

Cell lines derived from human tumors enable the properties of neoplastic cells to be analyzed under more controlled conditions than are possible in a clinical setting. This approach is especially important in the case of germ cell tumors in which histological complexity obscures their origins and the relationships between the various elements of which they may be composed. Interpreting the significance of alphafetoprotein (AFP) and human chorionic gonadotropin (BCG) production in patients with germ cell tumors illustrates these problems. Often the serum levels of these markers may be affected differently after therapy, indicating that they are produced by different cell types (Braunstein et aI., 1973). Also embryo­logical data would suggest that AFP (Gitlin and Boesman, 1967; Gitlin and Perricelli, 1970) is a marker of yolk sac carcinoma, and BCG (Midgley and Pierce, 1962) is a marker of choriocarcinoma. Neverthe­less , these proteins have been reported in patients in whom neither carci­noma has been recognized histologically (Javadpour et aI., 1978; Javadpour, 1980). Analysis of well-defined cell lines corresponding to the different components of these tumors may help to clarify existing his­tological classifications, which are based largely on morphology, and to

Peter W. Andrews: The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania

285 I. Damjanov et al. (eds.), The Human Teratomas© The HUMANA Press Inc. 1983

286 ANDREWS

provide new criteria for more sound diagnosis and assessment of methods of treatment.

In the mouse, cellular differentiation in teratocarcinomas has been shown to parallel differentiation during the early stages of embryogenesis; indeed, if the stem cells of these tumors are injected into blastocysts they may take part in normal development (for reviews, see Stevens, 1967; Hogan, 1977; Graham, 1977; Solter and Damjanov, 1979; Martin, 1980; Strickland, 1981). Since embryos are in short supply and provide little material for biochemical analysis, cell lines derived from murine teratocarcinomas offer a convenient alternative for investigating the mo­lecular processes of early mouse development. Likewise, we might sup­pose that cell lines derived from human teratocarcinomas will prove valu­able for studying early human embryogenesis. The existence of such a model system is especially important since experimentation with human embryos is nearly impossible, for ethical as well as logistical reasons. Moreover, the study of embryogenesis in other species cannot lead to a complete understanding of human development. Although one might ex­pect that the fundamental processes of embryonic cellular differentiation would be similar from one mammalian species to another, differences in detail are likely: divergent evolution may have led to changes in the or­ganization of the genome, to the loss of genetic elements, or to the estab­lishment of new ones that have no precise homo logs in other species . An example of the latter is the recent evolution of a locus coding for the hu­man placental isozyme of alkaline phosphatase (Goldstein and Harris, 1979). All such changes may have implications for comparative embryol­ogy. In any case, many morphological differences between species are apparent, even soon after cleavage begins (e.g., see Hamilton et aI., 1976).

In spite of these various and diverse reasons for studying human teratocarcinomas in vitro, relatively few detailed investigations have been made. In those studies that have been reported, investigators have often used the available experimental knowledge of murine teratocarcinomas as a starting point, assuming that the homologous murine and human tumor cells would exhibit similar properties: The pluripotent stem cells of mu­rine teratocarcinomas have been shown conclusively to be the morpho­logically undifferentiated embryonal carcinoma (EC) cells. These cells are characteristically small, with a high nucleus:cytoplasm ratio, have one or two prominent nucleoli, and grow in tight clusters when cultured in vitro. Developmentally, murine EC cells resemble cells of the inner cell mass or primitive ectoderm, and they share with these cells a number of biochemical properties such as the expression of certain embryonic an­tigens. Morphologically similar cells, again referred to as EC cells, have

HUMAN TERATOMA CELL LINES 287

also long been thought to be the stem cells of human teratocarcinomas (Dixon and Moore, 1952) although conclusive experimental data to con­firm or refute this concept are not yet available .

In this chapter, I shall attempt to review those studies of human teratocarcinoma-derived cell lines that have been reported but, being more familiar with them, I shall concentrate upon my own observations and those of my collaborators. We have studied many characteristics of a number of such cell lines and, in particular, have made a detailed investi­gation of one clonal line, 2102Ep, that seems to consist of human EC cells when grown under certain conditions. These EC cells have retained a limited capacity for differentiation in vitro. Our results suggest marked differences between human and mouse teratocarcinomas, and we feel that they help to clarify some of the confusing data that have previously been obtained.

Human Teratoma Xenograft Lines

Early attempts to derive cell lines from human germ cell tumors concen­trated on establishing explanted tumor biopsies as xenografts in immunosuppressed animal hosts (e.g., Pierce et aI., 1957; Pierce et al. 1958). The retransplantable xenografts established in this way included testicular embryonal carcinoma (EC) and choriocarcinoma passaged in the cheek pouches of cortisone-treated hamsters. Though these lines are no longer extant (Pierce, personnal communication), many new xenograft lines have been established, often in immunodeficient athymic (nulnu) mice, and these are listed in Table 1.

Retransplantable teratomas maintained as xenografts provide an op­portunity to study the histopathology of these tumors under more controlled conditions than would otherwise be the case with clinical bi­opsy material. For example, the study of a xenograft line that produces AFP has suggested new ways of interpreting the histology of seminomas and yolk sac carcinomas (Raghavan et aI., 1981). Xenografts have also been used to investigate the potential of various chemotherapeutic re­gimes (e.g., Verney et aI., 1959; Raghavan, 1980), and to test radiolabeled antibody in tumor-imaging techniques for locating tumor masses by external scintigraphy (e.g., Ballou et aI., 1979; Raghavan, 1980; Moshakis et aI., 1981). However, although much information may be gained from the study of xenografts, and indeed the capacity for differ­entiation of a cell line may be best seen when it has the possibility of forming three-dimensional structures, their maintenance and manipula­tion is inconvenient. Moreover, the growth conditions to which the tu­mors are exposed may vary with changes in the physiological state of the

288 ANDREWS

Table 1 Human Teratoma Cell Lines Established as Xenografts

Name Characteristics· References

ECCS EC Tveit et aI., 1980 HX36 MT, C Selby et aI., 1979 HX39 EC Raghavan, 1980; Raghavan et aI., 1980a, b;

Moshakis et aI., 1981 HX53 S, Y Raghavan, 1980; Raghavan et aI., 1980a, b, c;

Raghavan et aI., 1981 HX57 Y Raghavan, 1980; Raghavan et aI., 1980a,

b, c HX67 S, Y, EC Raghavan, 1980; Raghavan et aI., 1980a, b HX84 EC Raghavan, 1980; Raghavan et aI., 1980a,

b, c HX111 EC Raghavan, 1980 HXI12 Y Raghavan, 1980 HYST Y Shirai et aI., 1977 OE Y Yoshimura et aI., 1978; Hata et aI., 1980 TE Y Yoshimura et aI., 1978; Hata et aI., 1980 TI-I-JCK Y Yoshimura et aI., 1978; Hata et aI., 1980 EST-l Y Takeuchi et aI., 1979; Kaneko et aI., 1980 EST-2 Y Takeuchi et aI., 1979; Kaneko et aI., 1980

aEC, embryonal carcinoma; MT, malignant teratoma; C, choriocarcinoma (tera­toma, trophoblastic); Y, yolk sac carcinoma.

host, and the presence of host supporting tissues may confound biochemi­cal studies. Thus, for investigations aimed at characterizing the properties of the different elements of teratocarcinomas, cell lines adapted to growth in vitro, preferably clonal, and capable of producing tumors when in­jected into xenogeneic hosts, are the most useful experimental material.

Human Teratocarcinoma Cell Lines Established In Vitro

In recent years many cell lines derived from human germ cell tumors have been established in tissue culture (Table 2) . Mostly these lines are mor­phologically heterogeneous, suggesting that differentiation may be occurring in vitro but, in general, the cell types present have not been well-characterized and few lines have been cloned. When injected into immunodeficient animal hosts some lines produce tumors that are histo­logically consistent with EC. However, extensive differentiation has not been observed.

HUMAN TERATOMA CELL LINES 289

One line, PA-l (Giovanella et aI., 1974), derived from a malignant ovarian teratoma, was studied in some detail by Zeuthen et ai. (1980). When injected into athymic mice (nulnu), these cells in early passage gave rise to tumors that contained neuroepithelial elements and undifferentiated mesenchyme consistent with EC. However, in later passages the tumors were almost entirely composed of neuroepithelial cells . Some differentiation in vitro from small EC-like cells to larger, flat­ter epithelial cells was noted and it was suggested that this might repre­sent differentiation towards endoderm. The production of some AFP in aged cultures supported this notion. Pigmented cells were also noted.

In a comparative study of eight human cell lines derived from testic­ular teratocarcinomas, Andrews et ai. (1980) observed that three (1218E, Tera 2 and 577MF) were morphologically distinct from the other lines and also from murine EC lines in vitro. Of these only 577MF was tumorigenic in athymic mice, but the tumors produced were classified as carcinomas without the distinctive features of EC. Of the five other lines (Tera 1, SuSa, 833KE, 1156QE and 21 02Ep), cultures contained varying proportions of small undifferentiated cells, suggestive of EC cells, in ad­dition to other large, flatter cells. In athymic mice two of these lines (833KE and 2102Ep) produced tumors histologically consistent with hu­man EC, but no areas of differentiation were observed. Previously, Jewett et al. (1978) also observed some EC-like tumors derived from their subline of Tera 2.

To define the characteristics of human EC cells in vitro, Andrews et ai. (1982) made a more detailed study of one of their lines, 2102Ep. It had been established from a primary testicular germ cell tumor containing elements of EC, yolk sac carcinoma, and teratocarcinoma, but only EC has ever been observed in the tumors formed in athymic mice. Provided that cultures of 21 02Ep were passaged in vitro at a high cell density, uni­form populations of small undifferentiated cells with the morphological characteristics of EC cells could be maintained. Similar cultures could be re-established from the EC tumors of 2102Ep in athymic mice. Clones isolated from either the parental line or lines re-established from these tu­mors exhibited identical characteristics in vitro and also formed EC tu­mors in athymic mice. Therefore, 2102Ep was defined as a human EC cell line. A further observation indicated that these human EC cells re­tained some capacity for differentiation in vitro. If cultures were initiated at a low cell density, many cells differentiated into larger, flatter cells and the cultures became heterogeneous. This morphological change was also accompanied by changes in the expression of cell surface molecules, and this is discussed below.

290 ANDREWS

Table 2 Human Teratoma Cell Lines Established In Vitro

Cell line

577MRa 577ML" 577MP

833KE

1156QE

1218E

1242B 12550 2044L 2061H 2102EP'

2102ER' Huttke

LICR-LON HT-l LICR-LON HT-3 LICR-LON HT-5 LICR-LON HT-7 LICR-LON HX3917

Tumor Patient diagnosis

24 yr <3 TC 24 yr <3 TCa 24 yr <3 TCa

Tumori­genicity in xenogeneic

hosts References

Wang et aI., 1980 Wang et aI., 1980

Non-EC, Wang et aI., 1980; An-undiffer- drews et aI., 1980 entiated carcinomab

19 yr <3 EC, C, S, T Nude mice, Bronson et aI., 1978;

22 yr <3

23 yr <3

23 yr <3 22 yr <3 24 yr <3 19 yr <3 23 yr <3

23 yr <3 38 yr <3

20 yr <3 26 yr <3 24 yr <3 32 yr <3 40 yr <3

EC,C, S

EC, S

EC TC, S

EC TC

TC, Y

TC TC, S

EC,C,T EC, Y

EC, S, Y, T EC, Y

EC

EC Andrews et aI., 1980; Bronson et aI., 1980; Wang et aI., 1980

Andrews et aI., 1980; 'ang et aI., 1980

Andrews et aI., 1980; Wang et aI., 1980

Wang et aI., 1980 Wang et aI., 1980 Wang et aI., 1980 Wang et aI., 1980

Nude mice, Andrews et aI., 1980; EC 1982; Wang et aI.,

1980 Wang et aI., 1980 Evans, personal com­

munication; Nicholas, 1979; Avner et aI., 1981

Cotte et aI., 1981 Cotte et aI., 1981 Cotte et aI., 1981 Cotte et aI., 1981 Cotte et aI., 1981

(continued)

Biochemical Properties

Tables 3 and 4 indicate the range of the investigations of human teratocarcinoma-derived cell lines that have been reported. Some of these studies have concerned the production of markers that seem to be charac­teristic of certain cell types and have an established, or potential, value in

HUMAN TERATOMA CELL LINES 291

Cell line Patient

NEC-8 24 yr 0

Ovarian EC 63 yr S?

PA-l 12 yr S?

SuSa 30 yr 0 Tera 1 47 yr 0

Tera 2 22 yr 0

Table 2 (cant.)

Tumor diagnosis

EC

ECd TCe

TCY EC,T

EC, T

Tumori­genicity in xenogeneic hosts References

Hamster, Yamamoto et al., 1979 EC

Kimoto et al., 1975 Nude mice, Giovanella et al., 1974;

NE, UM' Zeuthen et al., 1980 Hogan et al., 1977 Fogh and Trempe, 1975;

Fogh, 1978; Jewett, 1978; Wang et al., 1980

Nude mice, Fogh and Trempe, 1975; EC Fogh, 1978; Jewett,

1978; Wang et al., 1980

Abbreviations: EC, embryonal carcinoma; C, choriocarcinoma; T, teratoma; TC, terato­carcinoma; Y, yolk sac carcinoma; NE, neuroectoderm; UM, undifferentiated mesenchyme.

"These lines were each derived from metastases (retroperitoneal, lung , and forehead , re­spectively) of the same patient. 577ML and 577MF were derived from autopsy specimens; pa­thology reports on these metastases are not available and TC is inferred from the pathology of the retroperitoneal lymph node metastasis (Bronson, personal communication).

"The tumors produced by this line in athymic mice were not consistent with EC. 'These lines were derived from the primary tumor and a retroperitoneal lymph node metas­

tasis of the same patient. dPeritoneal ascites from primary ovarian carcinoma. Reported as cysto-adenocarcinoma,

with multiple histological patterns and resembling EC. 'Derived from the ascitic fluid of a patient with recurrent malignant ovarian teratoma. Nude

mouse tumor reported to contain neurectoderm and undifferentiated mesenchyme, resembling embryonal variant of malignant teratoma.

fThe pathology of this tumor was described according to the British classification as a ma­lignant testicular teratocarcinoma, intermediate grade B with extensive necrosis. The patient's serum contained high levels of HCG.

the clinical management of these tumors: thus, HCG and AFP production have been studied since the consistent detection of large amounts of these proteins, which are generally taken as markers of choriocarcinoma and yolk sac carcinoma, respectively (with the reservation noted in the intro­duction), might indicate a cell line corresponding to these tumor types. Xenografted testicular choriocarcinomas (Pierce et al., 1958) did produce chorionic gonadotropin, whereas several studies of xenografted yolk sac tumors have shown that they produce AFP (Shirai et al., 1977; Yoshimura et al., 1978; Takeuchi et al., 1979; Hata et al., 1980; Kaneko

292 ANDREWS

Table 3 Biochemical and Functional Aspects of Human Teratocarcinoma-Derived Cell

Lines··b

Marker

HCG

AFP

CEA

Fibronectin

Collagen

Plasminogen activator

Alkaline phosphatase

Acid phosphatase

Lactate dehydrogenase

Studies of human teratocarcinoma­derived cell lines

3, 8

3,8,28

8

28

8

3,8,28

3, 5, 6, 8,28

5

3

Significance

Produced by fetal trophoblast (17); in GCT patients serum HCG correlates with troph­oblastic elements (14)

Produced by (visceral) yolk sac of embryo (10,12); in GCT patients AFP correlates with yolk sac carcinoma elements (25)

Found in fetal gut and produced by a variety of mostly endodermal tumors (13); found in serum of a few GCT patients (14)

Murine EC cells produce, but do not lay down, fibronectin (26, 27)

Murine EC cells have been reported to pro­duce type IV collagen; other forms may be produced upon differentiation (2)

Produced by the trophoblast and parietal endoderm of the early mouse embryo. Not produced by murine EC, but by PYS de­rivative cells (23)

Murine EC cells express high levels, which are reduced upon differentiation (7, 9)

Absent from murine EC cells; present in dif­ferentiated derivative (4)

LDH-5 observed to predominate in murine EC cells (15); LDH-l predominated in two xenografted human yolk sac tumors (24)

(continued)

et al., 1980) as well as other serum proteins known to be synthesized by the normal fetal yolk sac (Yoshimura et al., 1978; Hata et al., 1980).

Most of the in vitro cultures of human teratocarcinoma cells have not been reported to produce consistently large amounts of either HCG or AFP. Cotte et al. (1981) did note transient production of HCG while they were establishing several of their lines, but this property was subse­quently lost. Also Hogan (personal communication) has observed signifi­cant HCG production by SuSa, and considers that this line may represent a testicular choriocarcinoma cell line. In their study, Andrews et al. (1980) did detect traces of HCG in cultures of 833KE, 2103Ep, and their subline of SuSa and also traces of AFP in culture of 2102Ep, but produc-

HUMAN TERATOMA CELL LINES 293

Marker

Creatine kinase

High molecular weight glycoproteins

Metabolic cooperation with EC cells

Le-;tin (FBP and PNA) binding

Table 3 (cont.)

Studies of human

teratocarcinoma-derived cell lines Significance

3 Brain isozyme is expressed by murine EC cells (1)

19 High molecular weight glycopeptides were observed specifically in murine EC cells and embryos after pronase digestion (18)

21 In the mouse EC-EC and EC-ICM metabolic cooperation observed; no cooperation ob­served between EC and non-EC cells (20)

16 Murine EC cells bind more FBP and PNA than differentiated derivatives (II, 22)

"Abbreviations: AFP, alphafetoprotein; CEA, carcinoembryonic antigen; EC, embryonal carcinoma; FBP, L-fucose binding protein from Lotus tetragonolobus; GCT, germ cell tumor; HCG , human chorionic gonadotropin; LDH, lactate dehydrogenase (EC.l.I.I .27); PNA, peanut agglutinin; PYS, parietal yolk sac.

·References: I. Adamson et aI., 1977; 2. Adamson et aI., 1979; 3. Andrews et aI., 1980; 4. Avner et aI., 1977; 5. Avner et aI., 1981 ; 6. Benham et aI., 1981; 7. Bemstine et aI., 1973; 8. Cotte et aI., 1981 ; 9. Damjanov et aI. , 1971; 10. Dziadek and Adamson, 1971; II. Gachelin et aI. , 1976; 12. Gitlin and Perricelli , 1970; 13. Gold and Freedman, 1965; 14. Javadpour et aI. , 1978; IS . Lo and Gilula, 1980; 16. McIlhinney, 1981; 17. Midgley and Pierce, 1962; 18 . Muramatsu et aI., 1978; 19. Muramatsu et aI. , 1979a; 20. Nicholas et aI., 1978; 21. Nicholas , 1979; 22. Reisner et aI. , 1977; 23 . Sherman et aI. , 1976; 24. Takeuchi et aI. , 1979; 25. Talerman et aI. , 1980; 26. Wolfe et aI., 1979; 27. Zetter and Martin, 1978; 28. Zeuthen et aI., 1980.

tion was neither consistent nor sufficient to suggest that choriocarcinoma or yolk sac cells were predominant in their cultures. They suggested that variable, trace production of these markers could indicate sporadic differ­entiation of a stem cell into trophectodermal (HCG) or endodermal (AFP) elements.

Other secreted molecules, such as carcinoembryonic antigen (CEA) (Gold and Freedman, 1965; Talerman et aI., 1977), and pregnancy spe­cific ~I-glycoprotein, also known as SPI (Tartarinov et aI., 1974; Tartarinov , 1980) have been suggested as possibly useful tumor markers in teratocarcinoma patients. CoUe et al. (1981) found some production of CEA by several of their lines while they were being adapted to growth in vitro, but this ability was eventually lost. The significance of CEA pro­duction is unclear, but it could suggest some tendancy for differentiation, perhaps toward endodermal elements. SP-l production by the cell lines remains to be studied although it could be useful for identifying trophectodermal differentiation.

294 ANDREWS

Table 4 Cell Surface Antigen Expression by Human Teratocarcinoma Cell Lines

Studies of human

teratocarcinoma-Antigen derived cell lines Significance

HLA-A,B,C HLA and H-2 (the murine homolog) anti­gens are expressed by most somatic cells (8); but H-2 is not expressed by murine EC cells (2 , 6, 26)

-allogenic 7, 10, 12 determinants 22, 31

-monomorphic I, 2, 4, 7, 8 determinants 10, 11

HLA-Dr (Ia) I, 7 Ia antigens are not expressed by murine EC cells (13)

I3rMicrogiobulin I , 2, 7, 1O, I3rMicrogiobulin is a subunit of HLA­A,B,C, and H-2 D and K antigens as well as other antigens (e.g . , TLa); it is not present on murine EC cells (14)

12, 23 , 31

F9 7, 12, 22, 23

PCC4 7

Conserved murine embryonic antigen de­fined by a syngeneic antiserum and ex­pressed by murine EC cells (5, II)

Conserved murine embryonic antigen de­fined by a syngeneic antiserum and expressed by some murine EC cells (18)

Conserved murine embryonic antigen de-Antigen-I 27

SSEA-I I, 4 , 10, 25

SSEA-3 4,28

fined by a syngeneic antiserum and ex­pressed by murine EC cells (15. 20)

Monoclonal antibody-defined, conserved murine embryonic antigen expressed by murine EC cells (30)

Monoclonal antibody-defined, conserved murine embryonic antigen expressed by pre implantation murine embryos and not expressed by murine EC cells (28)

(continued)

Other biochemical and immunological markers have proved useful in analyzing murine teratocarcinomas and murine embryonic develop­ment, and so have also been applied to the study of human teratocarcinomas: thus Nicholas (1979) noted that two human teratocarcinoma cell lines (Tera I and Huttke) would undergo metabolic cooperation with murine EC cells although no cooperation was observed with murine non-EC cells or with other human cells (HeLa). As murine EC cells were observed to undergo metabolic cooperation only with other EC cells or similar embryonic cells, this suggested that these two human lines both contained a population of EC cells. Similarly, high molecular

HUMAN TERATOMA CELL LINES 295

Table 4 (cont .)

Studies of human

teratocarcinoma-Antigen derived cell lines

602-29 3

LICR.LON 4.1 25

HTI7 25

FIB75 25

Placental I, 3, 9 alkaline phosphatase

ECMA2 7

ECMA3 7

Significance

Monoclonal antibody-defined, human anti­gen coded by chromosome 12 and ex­pressed by most human cell including teratocarcinoma (3)

Conserved antigen expressed by murine EC cells and defined by a monoclonal anti­body (16)

Conserved antigen expressed by murine EC cells and defined by monoclonal antibody (16)

Antigen expressed by all cell lines from dif­ferentiated tissues (17)

Can be detected as a cell surface antigen by antisera and monoclonal antibodies (31). ALP activity is high in murine EC cells, but a specific placental locus does not ex­ist in mice (19 , 21)

Monoclonal antibody-defined antigen pres­ent on murine EC cells (24)

Monoclonal antibody-defined antigen pres­ent on murine EC cells (24)

References: J. Andrews et a!., 1980; 2. Andrews et a!., 1981 a; 3. Andrews et a!., 1981 b; 4. Andrews et a!. , 1982; 5. Artzt et a!. , 1973; 6. Artzt and Jacob, 1974; 7. Avner et a!. , 1981; 8. Barnstable et a!., 1979; 9. Benham et a!. , 1981 ; 10. Bronson et a!. , 1980; I J. Buc-Carron et a!., 1974; 12 . Carroll et a!., 1980; 13. Delovitch et a!. , 1978; 14. Dubois et a!., 1976; 15. Edidin et aI., 1971 ; 16. Edwards et aI., 1980; 17. Edwards , unpublished results , quoted by McIlhinney , 1981 ; 18. Gachelin et a!., 1977; 19. Goldstein and Harris, 1979; 20. Gooding et a!., 1974; 21. Hass et a!., 1979; 22. Hogan et a!. , 1977; 23. Holden et a!., 1977; 24. Kemler et aI., 1979; 25. McIlhinney et a!. , 1981 ; 26. Morello et aI., 1978; 27. Ostrand-Rosenberg et a!., 1977; 28 . Shevinsky et aI., 1982; 29. Slaughter et aI. , 1981 ; 30. Solter and Knowles , 1978; 3 I. Zeuthen et aI., 1980.

weight glycopeptides (Muramatsu et aI., 1979), high alkaline phospha­tase levels (Andrews et al. 1980; Cotte et al. 1981; Zeuthen et aI., 1980; A vner et aI. , 1981; Benham et aI., 1981), and low acid phosphatase lev­els (Avner et aI., 1981), and relatively high binding of Lotus tetragonolobus and peanut lectins (McIlhinney, 1981) have been ob­served in a variety of human teratocarcinoma cell lines. As these are all characteristics of murine EC cells, it was again suggestive that at least some of the human lines studied contained EC popUlations. On the other

296 ANDREWS

hand the isozymes of lactate dehydrogenase (LDH) and creatine kinase (CK) expressed by a number of lines (Andrews et aI., 1980) were dissimi­lar to those of murine EC cells (Adamson et aI., 1977; Lo and Gilula, 1980).

The three aspects of human teratoma cells that have received the most attention concern the expression of certain cell surface molecules, namely the major histocompatibility antigens, antigens cross-reacting with embryonic antigens of the mouse, and the enzyme alkaline phospha­tase (ALP). Because these molecules may be recognized immunologi­cally on the cell surface, they provide important tools for dissecting mixed cultures of cells and for isolating their component subpopulations by immunoselection techniques. The analysis of the expression of the cell surface molecules has indicated a number of probable differences be­tween the human and mouse teratocarcinoma systems.

Major Histocompatibility Antigens

Comparative studies between the expression of the major histocompatibility antigens by mouse and human teratocarcinoma cells are relatively straightforward as the two homologous genetic systems are well understood (for a review, see Barnstable et aI., 1979). The H-2K and D antigens have been shown to be absent from murine EC cells (e.g., Artzt and Jacob, 1974; Morello et aI., 1978; Andrews and Goodfellow, 1980), but these antigens do appear upon differentiation in culture (e.g., Nicholas et aI., 1975; Knowles et aI., 1980). Also, murine EC cells do not express the H-2K- and D-associated molecule, 132-microglobulin (Dubois et aI., 1976), nor the Ia antigens (Delovitch et aI., 1978). Whether or not H-2 antigens are expressed by early mouse embryos re­mains controversial (for a review, see Wiley, 1979) and will not be dis­cussed further here.

In humans, information concerning early embryos is non-existent, although it is known that the expression of HLA (the human homolog of the murine H-2 antigens) on term placenta is, at most, very low (Goodfellow et aI., 1976). Also, some gestational choriocarcinoma cell lines lack HLA-A, -B, and -C antigens (Trowsdale et aI., 1980). The ab­sence of these antigens from the trophoblast may in part account for the lack of maternal rejection of the fetus.

In studying human teratocarinoma-derived cell lines, it has been as­sumed that the human EC cells, as in the mouse, would also lack the ma­jor histocompatibility antigens and I3rmicroglobulin. Because signifi­cant levels of I3rmicroglobulin were detected on Tera 1 cells, but not on

HUMAN TERATOMA CELL LINES 297

Tera 2, Holden and his colleagues (1977) regarded Tera 2 as an EC line and Tera 1 as a differentiated line derived from the original EC tumor. Similarly, Hogan et al. (1977) found a small proportion of HLA-A,B,C­positive cells in their line , SuSa, and suggested that these cells repre­sented differentiated derivatives from HLA-negative stem cells .

More recently, A vner et al. (1981) reported that 100% of the cells in cultures of the PA-l ovarian line were HLA-positive . Also, Andrews et al. (1980) noted that eight different testicular teratocarcinoma cell lines (including Tera 1, Tera 2, and SuSa) each contained variable porportions of HLA-A,B,C-positive and 132-microglobulin-positive cells, ranging from about 10% in Tera 2 to greater than 90% in 833KE. The almost 100% positive cells in the 833KE cultures was notable since this line pro­duced pure EC tumors in athymic mice. Moreover, in all of these human teratocarcinoma cell lines HLA and I3rmicroglobulin immunofluor­escence was quite weak when compared to other human cell types, and it was suggested that the apparent existence of HLA-positive and -negative subpopulations in some lines could be an artifact resulting from low aver­age expression, comparable to the limits of sensitivity of the assay. Fur­ther, analysis of the same lines by flow cytofluorimetry (Andrews et al., 1981a) supported this explanation. HLA-A, -B , -C and I3rmicroglobulin are also expressed by the 2102Ep human EC cell line, and significant changes in expression were not seen upon differentiation at low cell den­sity (Andrews et al., 1982). Thus, the available evidence indicates that human EC cells differ from murine EC cells by expressing, at low levels, antigens of the major histocompatibility complex (HLA-A, -B, -C) and I3rmicroglobulin. Consistent with this is the observation that somatic cell hybrids of murine EC cells with human cells also express HLA-A, B, C provided that they retain chromosome six (Benham et aI., 1983).

As in the mouse, none of the human lines has been shown to express Ia (HLA-D-related) antigens (Andrews et al., 1980) . This is also true of 2102Ep cells, which fail to bind a monoclonal antibody, DA-2, recognizing a monomorphic determinant of the human HLA-D-related antigens (Brodsky et al. 1979).

Embryonic Antigens

Antisera produced by xenogeneic immunization (followed by absorption) (Edidin et aI. , 1971; Gooding and Edidin, 1974) and by syngeneic immu­nizations (see review by Gachelin, 1978) have been used to detect a series of embryonic antigens on murine EC cells. Probably the most extensively studied of these antigens are F9 (Artzt et aI., 1973; Buc-Carron et aI.,

298 ANDREWS

1974) and PCC4 (Gachelin et al., 1977) , defined by antisera produced by the syngeneic immunization of 129 mice with F9 and PCC4 murine EC cells, respectively . Anti-F9 sera were found to react with all murine EC cells tested and with murine embryos after the morula stage. Anti-PCC4 sera, after absorption with F9 cells, defined another embryonic antigen(s) temed PCC4 and showed reactivity with a more restricted group of mu­rine EC cells. Several cell surface molecules expressed by murine EC cells and embryos have now been defined by monoclonal antibodies (Solter and Knowles, 1978; Stern et al., 1978; Goodfellow et al., 1979; Kemler et al., 1979), and these have been the subject of many recent studies (for review, see Solter and Knowles, 1979).

Ostrand-Rosenberg et al. (1977) were among the first to show that human teratocarcinoma cells share antigenic determinants with murine EC cells and embryos. They observed reactivity of Tera 2 cells with the rabbit anti-murine teratocarcinoma (402AX) serum of Edidin and his col­leagues (1971). In addition, they found that serum from four out of six patients with teratocarcinomas sometimes showed specific reactivity with the murine teratocarcinoma cell line 402AX. Teodorczyk-Injeyan et al. (1980) also observed that sera from some teratocarcinoma patients react with murine teratocarcinomas.

Antisera defining the F9 and PCC4 antigens have similarly been found to react with a variety of human teratocarcinoma cell lines. In early studies of murine teratocarcinoma cells an inverse relationship between the presence of the F9 antigen(s) and the H-2 antigens was observed (Nicholas et al., 1975); undifferentiated EC cells were H-r 1F9 + whereas their differentiated derivatives were found to be H-2+ 1F9 -. Thus, when Holden et al. (1977) and Hogan et al. (1977) showed an ap­parently analogous relationship between the expression of the F9 and HLA antigens by Tera 1, Tera 2, and SuSa cells, it seemed that a similar­ity between human and mouse EC cells might hold. However, more re­cently A vner et al. (1981) found HLA + 1F9 + cells in cultures of Tera 1. In the study by Zeuthen et al. (1980), PA-l cells were found to be F9-negative although they were thought to be stem cells with some poten­tial for further differentiation; some of these cells were shown to react with antisera defining the PCC4 antigen(s).

The expression of several murine embryonic antigens defined by monoclonal antibodies have now been studied on human teratocarcinoma cell lines . These include three, ECMA-2 and ECMA-3 (Kemler et al., 1979) and SSEA-l (Solter and Knowles, 1978), which resemble the orig­inal description of the F9 antigen(s) in their cellular distribution. In a study of five human teratocarcinoma cell lines (Tera 1, Tera 2, SuSa, PA-l, and Huttke), Avner and colleagues (1981) were unable to detect

HUMAN TERATOMA CELL LINES 299

either ECMA-2 or ECMA-3. On the other hand, Andrews et aI., (1980) observed variable expression of SSEA-l by seven out of eight lines that included Tera 1, Tera 2, and SuS a; only the 577MF cell line, which does not express any EC-like characteristics, was found to lack SSEA-l com­pletely. However, several lines, particularly two sublines of 833KE, that morphologically most resemble EC cells, were SSEA-I-negative in most assays.

These observations were clarified by further studies of the clonal hu­man EC cell line 2102Ep (Andrews et aI., 1982). Provided cultures were maintained under high density conditions that resulted in pure EC-like populations, no SSEA-l could be detected. It was concluded that human EC cells differed from murine EC cells by lacking SSEA-l determinants. SSEA-l expression was only observed when these EC cells underwent morphological differentiation at low cell density. The variable expression of SSEA-l by other human teratocarcinoma cell lines in culture is proba­bly related to spontaneous differentiation of the type seen in low density cultures of 2102Ep. The nature of this differentiation and the equivalent embryonic cell types of the SSEA-l- human EC stem cell and its SSEA-l + derivative remain to be elucidated. However, some further in­formation was obtained from the study of another murine embryonic anti­gen, SSEA-3, recently defined by a new monoclonal antibody raised against pre-implantation mouse embryos (Shevinsky et aI., 1982).

SSEA-3

Whereas SSEA-\ is expressed on mouse embryos after the morula stage, and on inner cell mass cells, SSEA-3 is an antigen found on unfertilized eggs, zygotes, and early cleavage stages of the mouse; it is absent from the primitive ectoderm. SSEA-3 is also absent from murine EC cells. Nevertheless, when the reactivity of the anti-SSEA-3 antibody was tested on a panel of 80 different human tumor cell lines, most teratocarcinoma­derived lines were found to express this antigen. Exceptions were Tera 2 and 577MF, lines that probably no longer contained any human EC cells. No other type of tumor tested reacted with this antibody, although periph­eral red blood cells were also found to be SSEA-3 positive.

These observations led to a more detailed analysis of SSEA-3 ex­pression in the human EC cell line, 2101 Ep (Andrews et aI., 1982). In high density, EC-like cultures SSEA-3 was expressed strongly and most cells were scored positive by immunofluorescence. When differentiation at low cell density occurred, there was a reduction in SSEA-3 expression, which coincided with the appearance of SSEA-\. This was investigated

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further by irnmunoselection using complement-mediated cytotoxicity. These experiments suggested the presence of SSEA-l-/SSEA-3+, SSEA-l + /SSEA-3 +, and SSEA-l + /SSEA-r cells in low density cul­tures of 2102Ep, and that morphological differentiation was accompanied by a change in surface antigen phenotype, from SSEA-USSEA-3+(EC) cells to SSEA-l + /SSEA-3 - (non-EC, differentiated) cells. Immunoper­oxidase staining of tumor specimens from teratocarcinoma patients and from mice carrying xenografts has confirmed that human EC cells are SSEA-l-/SSEA-3 + (Damjanov et aI., 1982).

Although their molecular structures remain to be fully established, the available data suggest that simple conversion of SSEA-3 to SSEA-l (e.g., by a glycosylation) is unlikely to explain the change from the SSEA-l-/SSEA-3+ to the SSEA-l+/SSEA-r phenotype: the SSEA-l antigenic determinant is a fucosylated lactosamine related to the IIi blood group carbohydrate (Hakomori et aI., 1981; Gooi, 1981), although its precise structure is not yet known. It exists in the cell surface as a glycolipid and also as molecules of very high molecular weight, perhaps proteoglycans (Andrews et aI., 1981c). By immunoprecipitation no gly­coproteins carrying the SSEA-l determinant have been observed (unpub­lished results). SSEA-3 is also a carbohydrate determinant but seems to have a structure radically different from SSEA-l (Hakomori and Knowles, personal communication) . It appears to exist in the cell surface as glycolipid, but immunoprecipitation has revealed a number of glyco­proteins that also carry this determinant (Shevinsky et aI., 1982). These differences between the SSEA-l and SSEA-3 molecules suggest that the differentiation of 2102Ep human EC cells observed in low density cul­tures must involve more fundamental changes in cellular physiology than the activation of a single glycosyltransferase.

Alkaline Phosphatase (ALP)

ALP is a useful marker of differentiation in the mouse (Damjanov et aI. , 1971; Bemstine et aI., 1973; Solter et aI., 1973; Martin and Evans, 1975). Found at high levels in ICM and primitive ectoderm cells of mouse embryos, as well as in murine EC cells, its activity is much re­duced in endoderm and other differentiated cells . Thus, it is of some in­terest to investigate this enzyme in human teratocarcinomas. However, direct comparison between the mouse and human systems is difficult be­cause of differences in the number of genetic loci that code for ALP (Goldstein and Harris, 1979). In the mouse, two loci code for distinct in­testinal and liver/bone/kidney isozymes; the isozyme present in embryos

HUMAN TERATOMA CELL LINES 301

and teratocarcinomas is apparently coded by the liver/bone/kidney ALP locus (Hass et aI., 1979). Homologous loci also exist in the human genome, but recent evolution has given rise to a third locus coding for placental ALP, an isozyme normally found in the fetal trophoblast (Fishman, 1974; Goldstein et aI., 1980a).

An additional point of interest with ALP is that it is a cell surface protein that can be detected immunologically as a cell surface antigen, as well as by virtue of its enzymatic activity. Monoclonal antibodies that specifically detect the different human isozymes are already available (Slaughter et aI., 1980, 1981; Harris, personal communication). This as­pect of ALP increases its potential usefulness as a cell marker in teratocarcinomas, especially if different cell types can be shown to ex­press different isozymes; for example , placental ALP might be an indica­tor of trophectodermal (choriocarcinoma) differentiation.

High levels of ALP have been detected in the human terato­carcinomas that have been investigated. On the basis of heat stability and inhibition studies, it has been shown that most of the activity is of the liver/bone/kidney form, but a variable proportion is of the placental form. However, in their study of eight lines, Benham et aI., (1981) observed some differences between the standard placental isozyme of term placenta and the similar form found in teratocarcinomas. These differences con­cerned inhibition by L-Ieucine and phenylalanylglycyl glycine , the heat­stable placental enzyme being stronly inhibited by phenylalanylglycyl glycine and only weakly by L-Ieucine, whereas the reverse was true for the heat-stable, placental-like form in teratocarcinomas. Similar variants of the placental enzyme have been detected, ectopically in other tumors, and also at a low level in some normal tissues, notably testes (Goldstein et aI., 1980b; Chang et aI., 1980). This leads to the possibility that the placental-like enzyme in these cell lines, derived from testicular teratocarcinomas, represents continued expression of a form found in the human testis, or that it is ectopic expression as found in other tumors, or that it represents a form expressed in some cells of the early human em­bryo. These possibilities are not mutually exclusive, and it is possible that the placental-like form could be the product of a previously unrecognized locus that cross-reacts with antisera and monoclonal antibodies that dis­tinguish placental alkaline phosphatase from the liver/bone/kidney and in­testinal isozymes. Using a monoclonal antibody reactive with placental ALP, it was found that several human teratocarcinoma cell lines contain subpopulations of cells expressing quantitatively different amounts of the placental-like isozyme (Benham et aI., 1981). Also, high density 2102Ep EC cells express a small amount of the placental alkaline phosphatase an­tigen, but the possibility remains that a few of their differentiated deriva-

302 ANDREWS

tives express rather higher levels (Andrews et aI., 1982). None of these observations of placental-like ALP correlated with the production of sig­nificant amounts of HCG that would suggest trophectoderm differentia­tion .

Conclusion

Investigations of cell lines derived from human teratocarcinomas are still in their infancy, despite their potential interest for both clinical scientists and developmental biologists. Although many such cell lines have been reported, few have been studied in detail and even fewer have been cloned. Also, there have been few attemps to relate the cell types seen in vitro to those observed in human tumors in situ. Because of this and be­cause many studies have considered different parameters and different cell lines, it is often difficult to interrelate their results. There is still no consensus as to the characteristic properties of the various cell types that occur in these tumors, or to the identity of the stem cell. Also, several studies seem to suggest that the properties of human and mouse teratocarcinoma cells are not as similar as might have once been ex­pected. Against this background, our own results provide new criteria for identifying the human EC cell, the putative stem cell of human teratocarcinomas, and suggest a working hypothesis to rationalize the various results obtained so far.

None of the human teratocarcinoma cell lines so far studied, either in in vitro culture or as xenografts, seem to possess a capacity for extensive differentiation, despite some morphologically heterogeneous cultures. Even in the case of the limited differentiation seen in lines such as 2102Ep or PA-l, it has so far been impossible to identify the derivative cell types. It may be thought that it is therefore premature to define the characteris­tics of human EC cells, especially if it is considered that EC cells should be defined as the pluripotent stem cells of human teratocarcinomas, as they have proved to be in the mouse. However, the term "embryonal car­cinoma" is primarily a description used by pathologists, and although EC cells have long been thought to be the stem cells of human germ cell tu­mors (Dixon and Moore, 1952) this has never been proved. Since the term EC is actually rather imprecise it becomes important to provide more objective criteria than simple morphological description. It is in this sense that we have used the 2102Ep cell line (derived from a human germ cell tumor, and capable of producing in athymic mice, tumors consistent with the histological description, embryonal carcinoma) to characterize human EC cells as expressing the cell surface antigen SSEA-3 and not SSEA-l, whereas murine EC cells express SSEA-l and not SSEA-3.

HUMAN TERATOMA CELL LINES 303

Another difference between human and mouse testicular teratocarcinomas may be considered here, namely the frequent occur­rence of choriocarcinomas (indicating trophectodermal differentiation) in humans but not in mice. The absence of choriocarcinomas in the murine tumors correlates with the observation that murine EC stem cells are developmentally equivalent to embryonic cells of the inner cell mass and primitive ectoderm, cells that have lost the capacity to differentiate into trophoblast (Rossant, 1977). The corollary of this is that the stem cells of human teratocarcinomas are developmentally equivalent to earlier embry­onic cells that still retain the capacity for trophectodermal differentiation. The observation that human EC cells are SSEA-l-/SSEA-3 + (the sur­face antigen phenotype of cleavage stage mouse embryos), whereas mu­rine EC cells are SSEA-l + /SSEA-r (the surface antigen phenotype of murine primitive ectoderm cells) is consistent with this hypothesis. Also we have recently found that human EC cells do not synthesize fibronectin, but differentiate into cells that do (Andrews unpublished ob­servations); murine EC cells have been shown to synthesize fibronectin (Wolfe et al., 1979).

Of course , we do not know how SSEA-l and SSEA-3 are expressed on human embryos , nor can we yet assess the significance of human EC cells expressing HLA-A, -B, -C, and ~2-microglobulin. Nevertheless, the available data do help to define the human EC cell, no matter to which embryonic cell it is finally to correspond. Once cultures capable of exten­sive differentiation are obtained, it will then be possible to establish whether human EC cells, so defined, are the stem cells or whether some other cell type possesses this capacity in human teratocarcinomas.

Acknowledgments

I should like to thank my colleagues and especially Drs . Barbara Knowles, Davor Solter, David Bronson, Ivan Damjanov, Wolter Oosterhuis, Peter Goodfellow, and Frances Benham for their help and support.

This work was supported in part by grants CA-18470 and CA-29894 from the National Institutes of Health, and grant 1860 from NATO.

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