Int . J. Cuncer: 42, 261-266 (1988) #j&@ Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer \, . - @ 1988 Alan R. Liss. Inc.

SKIN PAPILLOMAS AND OTHER NEOPLASMS INDUCED BY MURINE SARCOMA VIRUSES IN MID-GESTATION-INFECTED MICE Lorenzo R o s s ~ ' ~ ~ , Ottavia BARRIERI', Gloria CAPURRO' , Cinzia AIELLO~ , Tiziana RUZZON' and Olimpio MURATORE* ' Laboratov of in vivo Carcinogenesis, Institute of Clinical and Experimental Oncology, University of Genoa and Istituto Nazionale per la Ricerca sul Cancro. viale Benedetto XV, 10. 16132 Genoa; and 'Institute of Microbiology, University of Genoa, 16132 Genoa, Italy.

During extensive investigations on the effects of onco enic retroviruses in developing rodents, the ability of MS& to mount a neoplastic response in CD-I Swiss mouse embryos was determined. By infecting the animals directly in utero at selected stages of post-implantation development, we de- tected a uliar reaction of the embryonal tissues to certain MSVs: WE mice were exposed to KiMSV at mid-gestation, the newborn developed characteristic tumors, in addition to mesenchymal cell sarcomas, not induced in fetuses and neo- nates. These included pulmonary alveologenic tumors and skin papillomas and were seen in mice infected on days 8 and 10 of pregnancy, roughly corresponding to 15 and 35 somites, respectively. To determine the specificity of these events, other 8- and IMay-old embryos were infected with retrovi- ruses of the same or different families. HaMSV and MoMSV also induced mesenchymomas and a low incidence of skin papillomas (10% and 15% compared to 40% in the KiMSV group) but not pulmonary tumors. In contrast, FBRMSV was inactive in this respect and only osteogenlc sarcomas were detected in the offspring. Infecting the embryos on day 7 of pregnancy produced no tumors. Later infections (in 15-day-old fetuses and neonates) mainly induced mesenchymal sarcomas. No congenital malformations were detected in the embryos exposed to MSV during organogenesis, although some abor- tions and resorptions were seen.

An important insight into the mechanisms of prenatal carci- nogenesis is the finding that certain proto-oncogenes, homol- ogous to the transforming genes of murine sarcoma viruses (MSV), are developmentally regulated in mammalian embryos (Muller et al . , 1982; Slamon and Cline, 1984; Propst and van de Woude, 1985). The meaning of their presence in these organisms is not known but circumstantial evidence suggests that they may play a central role in various embryonic pro- cesses, including regulation of cellular differentiation and pro- liferation. When affected by specific mutations, these cellular elements (now called c-onc) are widely regarded as key factors in the inception and progression of spontaneous and induced neoplasms of many types (Duesberg, 1985; Weinberg, 1985; Mareel and van Roy, 1986).

Since the genome of the known MSV contains DNA se- quences apparently transduced from host cells (Bishop, 1985; Duesberg, 1987), we decided to probe the question of whether and how mid-gestation mouse embryos react to the carcino- genic stimuli carried by MSV. There are at least 2 reasons why such an investigation is of interest in the field of devel- opmental oncology. The first is that, in rodents, mid-gestation stages are characterized by fundamental morphogenetic changes, including the appearance of organ and tissue anlages. Our approach could therefore provide a useful model system to analyze the origins of tumors in early life. The second reason concerns the well-known fact that post-implantation rodent embryos are remarkably resistant to the induction of tumors by transplacental chemical carcinogens before the pla- centa is formed; aborted and malformed fetuses are frequently detected instead in pregnant females after such exposures (No- mura, 1974; Brent, 1977). The determination of whether or not the same phenomenon is also associated with oncogene- carrying retroviruses could have considerable implications in understanding the relationship between teratogenic and carci- nogenic events in developing rodents.

Data on carcinogenic activity of KiMSV and MoMSV in fetal and in newborn mice have already been published (Rossi et al . , 1985). We now describe the histopathological findings of an experiment dealing with the infection of 7- to 10-day-old mouse embryos with several strains of MSV. We have found that the induction of 2 types of neoplasm, namely skin papil- lomas and lung tumors, depends on the virus and on the day of pregnancy at infection.

Earlier work on the sensitivity of mouse embryos to carcin- ogens during early organogenesis have been limited to occa- sional observations made with conventional trans-maternal chemical carcinogens and intraembryonal injections of murine leukemia viruses (Spatz and Laqueur, 1967; Jaenisch, 1980).

MATERIAL AND METHODS

Mice CD-I outbred Swiss mice were purchased from Charles

River (Como, Italy) and maintained in our animal facility. At 7 to 10 weeks of age, groups of 3 females to a male were housed together in the evening and examined the following day for copulation plugs which, if present, determined day 0 of pregnancy. Viruses

The viral stocks of Kirsten (KiMSV), Harvey (HaMSV) and Moloney (MoMSV) strains of murine sarcoma viruses were prepared essentially as described (Rossi et al . , 1985) and dilut9,Qo the working concentration of 1 X l@.9, 1 X 106.' and 1 x 10 focus-forming units per ml, respectively. Thc murine osteosarcoma virus (FBRMSV) was a kind gift from Dr. J. Schmidt (Institute for Pathology, Neuherberg, FRG) and is now replicated in our laboratory. Its estimated concentration was about 1 X lo3.' focus-forming units per ml. The viral preparations were preserved at -80°C. The quantity of virus injected yielded not less than SO% of apparently healthy new- born in preliminary studies. Experimental procedure

On days 7, 8 or 10 of pregnancy, groups of 5-6 females were anesthetized (Nembutal) and laparotomized and the uter- ine horns were carefully externalized. Mid-gestation embryos were infected by micro-injection of a 0.5-1.0 p1 of each virus preparation directly through the uterus wall into the ventral third of the decidual swelling. FBRMSV was not tested on day 7 of embryonal growth. Appropriate control groups received the same volume of virus-free medium. At 15 days of preg-

%o whom reprint requests should be sent.

Abbreviations: MSV, murine sarcoma viruses; KiMSV, HaMSV, MoMSV and FBRMSV, Kirsten, Harvey, Moloney and murine osteosar- coma strains of MSV respectively.

Received: August 26, 1987 and in revised form January 12, 1988.

262 ROSS1 ET AL.

TABLE I - EXPERIMENTAL GROUPS AND DESIGN

N* Late pregnancy' - Mid-gestation'

7 8 10 15

Number of embryos injected: A ents and concentration3

k M S V 1 X 104.9 52 205 89 58 41

MoMSV 1 x lo3.* 79 103 31 65 66 34 39 31 18

HaMSV 1 x lo6.' 57 68 42 85 54

FBRMSV 1 x ld.* - Virus-free medium4 55 73 49 - -

Volume injected (pl): 0.5-1 1 1 10 20 'Day of pregnancy.-*N=Neonates less than 24 hr old.-3Focus-forming unit~/ml.-~The virus-free medium was composed of

Dulbecco's modified essential medium. 5% calf serum, glutamine (2 mM) and gentamicin (10 ml/liter).

TABLE I1 - SUMMARY (>I- ' I H t TUMORS FOUND IN CD-I MICE INFECTED WITH THE RETROVIRL'SES IOMSV. Ha.MSV. MoMSV AND FBRMSV DURING SELECTED STAGES OF EMBRYONAL DEVEL0PMt"I' OR AT BIRTH 1:VAI.IJATIONS WEKE MADE WHEN THF ANIMALS REACHED 20 WEEKS OF AGE -

Period of development

KiMSV HaMSV MoMSV FBRMSV KiMSV HaMSV MoMSV FBRMSV Late pregnancy and neonatal Mid-gestation'

Number of animals at risk2 202 97 104 53 94 115 111 46

Tumors at any site 54 23 1 1 13 23 69 78 28 Mesenchymal-cell sarcomas 25 14 6 133 14 68 67 283 Vascular tumors of the brain 4 2 0 0 1 1 2 3 0 Lung tumors 10 0 0 0 0 0 0 0 Thymic lymphomas 0 6 4 0 0 1 8 0 Skin DaDillomas 28 4 3 0 0 0 0 0

Number of animals with:

'Animals evaluated were infected on day 8 or 10 of embryogenesis.-2Number of animals which survived to at least I week of age.-3The tumors found in these groups were osteogenic sarcomas

n

66

HOHSV FBRHSV

100

1C

0

40

20

0

FIGURE 1 - Percentage of CD-1 offspring affected by tumors (empty bars, total tumors; hatched bars, mesenchymal cell sarcomas, or os- teogenic sarcomas in the FBRMSV groups) within the first 20 weeks of life. The number of mice at risk is reported at the top of each bar and the viruses used and the day of pregnancy on which they were in- jected at the bottom. N, newborn.

nancy, infection was carried out by micro-injecting 10 p1 of virus into the abdominal cavity of each fetus. Instruments needed for these intra-embryonal injections have been de- scribed (Rossi et al., 1985). When all the implants of a female

placed one to a cage and allowed to deliver their young. Newborns were counted and examined for the presence of external abnormalities. None of them had visible malforma- tions and the litter size was then adjusted to a maximum of 8. Other groups of newborns were infected by single i.p. doses of 20 p1 of each virus within the first 24 hr of life. In all, there were therefore 22 experimental groups as indicated in Table 1. The groups of KiMSV and MoMSV treatments on day 15 of pregnancy and in neonates are a new addition complementing previous data. In separate experiments not detailed here the Moloney murine leukemia virus (the helper in our viral stocks) was injected into mid-gestation and fetal mice and found to induce only hematopoietic neoplasms in the offspring. This rules out the possibility that the leukemic agent is the primary determinant o f other tumor types detected in our approach. Tumor histology

The offspring were checked daily for health conditions and for the appearance and growth of cutaneous nodules. These were counted and measured once a week. Animals found dead or killed when moribund were necropsied, then the diseased organs and tissues were fixed in 10% formalin and routinely stained with hematoxylin and eosin for histological examina- tion. When necessary, histochemical methods were also em- ployed to confirm diagnosis. The animals that survived to more than 20 weeks of age were apparently normal or had small skin papillomas. We decided to keep them alive in order to determine the biological characteristics of the neoplasms eventually arising late in life. Fisher's exact test was used for statistical significance.

RESULTS

The infection of 8-, 10- and 15.-day-old CD-1 Swiss mouse embryos, as well as of neonates, with the acutely sarcoma- were injected, the peritoneum and abdominal skin were su-

tured separately (Dexon, Danbury, CT), and the animals were genic retroviruses KiMSV, HaMSV, MoMSV and FBRMSV

TUMORS INDUCED BY MSV IN MOUSE EMBRYOS 263

FIGIJRE 2 - Tumors produced in CD-1 mice by direct treatment of 8- or 10-day-old embryos with KiMSV or FBRMSV. (a) Gross appearance of squamous papillomas in a 6-week-old male. (b) Light microscopy of one of these tumors showing morphology of squamous papillomas and mild intradermal infiltration. (c) Osteoid tumor which was attached to dorsal vertebral spina of a 10-week-old female. The histology is that of a periosteal sarcoma with abundant cartilaginous and osteoid trabeculae. In between are osteoblast-like elements embedded in a fibrous stroma. (4 Well-differentiated alveologenic adenocarcinoma affecting the lung of a 25-day-old male. The tumor-free portion is at the upper left. Sec- tions stained with hematoxylin and eosin.

caused the appearance of various types of tumor (Table 11). In contrast, none of the mice infected on day 7 of embryogenesis, nor of the control groups that received virus-free medium (of which more than 90% and 80% were alive after 1 week of age respectively), had tumors after more than 50 weeks of obser- vation; these are not included in Table 11.

The effects of the viruses were evaluated when the animals reached 20 weeks of age, at which time less than 50% survi- vors remained in most experimental groups. Neoplasms oc- curred mainly in the period between 1 and 10 weeks of age; the incidence was dependent on the virus and the age at infection, and independent of sex and litter size at birth. Perinutal mice are more sensitive to the carcinogenic effects of MSV than mid-gestation embryos

On the average HaMSV, MoMSV and FBRMSV raised the incidence of tumors from 11-24% to 60-70% following injec- tion during organogenesis or at perinatal stages, respectively (Table 11). Such a trend is also documented in Figure 1 show- ing the incidence of tumors per day of infection. The percent- age of animals affected by tumors was 32 % or lower when the infections were performed on day 8 or 10 of pregnancy and

57% or higher when 15-day-old embryos or neonates were infected. In contrast, with the Kirsten virus the average inci- dence of tumors remained the same whether mice were in- fected at mid-gestation or later developmental stages (27 % and 24% respectively) (Table 11). In this case, however, there were more offspring with tumors among those infected on day 10 of embryogenesis than in any other group (62% compared to 12%, 45% and 4% in the 8- and 15-day-old embryos and in the neonates respectively) (Fig. 1).

Most tumors found in our study were mesenchymal-cell sarcomas in which spindle-shaped cells predominated. Those induced during organogenesis, however, had a more primitive pattern and were frequently made up of tissues in which glandular structures and rhabdomyosarcoma cells were recog- nized. There was no indication that infecting mid-gestation embryos changed the target site and the multiplicity of these sarcomas per animal compared to the groups infected at birth, suggesting that the potentialities of the competent target cells are already restricted to specific organs and tissues as early as day 8 of embryogenesis.

Unlike the other viruses whose carcinogenic activity ap- peared directed towards a variety of target cells, FBRMSV

264 ROSS1 ET AL

TABLE 111 - FREQUENCY OF SKIN PAPILLOMAS IN CD-1 MICE INFECTED WITH MSV DIRECTLY IN UTERO AT MID- GESTATION

Day of pregnancy at infection 8 I0

Incidence' numbed max. diam. Incidence' numbed max. diam.

KiMSV 2.8(4/142) 1.7(1-3)' 9.5(8-10)' 40.0(24/60) 3.8(1-11)* 7.7(3-15)2

MoMSV 0(0/84) - - 15.0(3/20) 1.6( 1-2) lO(7-13) HaMSV 0(0/57) -

FBRMSV 0(0/24) - - 0(0/29) - -_

virus injected Average Average Average Average

animal (mm) animal (mm)

- 10.0(4/40) 1.7(1-3) 5.7(2-10)

'Percentage. Figures in brackets show the number of offspring with skin papillomas/total number of offspring at risk.- 'Numbers in'braccets represent the range.

induced only osteogenic sarcoma, a tumor originating from osteoblasts and commonly localized in the limbs and some- times in the ribs or the vertebral spina (Fig. 2).

Other neoplasms induced in our study, including endothelial- cell sarcomas of the brain and thymic lymphomas, were roughly the same, according to histology, incidence, and la- tency, as those described in a previous report (Rossi et al., 1985). The former were uncommon in the groups treated at mid-gestation but relatively abundant in those infected on day 15 of intra-uterine life, and were mainly associated with KiMSV (Table 11). Vascular telangiectasia of the brain and vascular ruptures and enlargement of the spleen were fre- quently observed in the KiMSV-, HaMSV-, and MoMSV- but not in the FBRMSV-infected mice.

f i e interaction of MSV with mid-gestation CD-1 mice yields skin papillomas

Unexpectedly, at about 3 weeks of age some of the young which were infected with KiMSV, HaMSV, and MoMSV at mid-gestation began to be affected by squamous papillomas of the skin. These lesions presented mainly as pedunculated, single or multiple neoformations, diversely distributed over the surface of the body, and were histologically composed of squamous cells at several stages of differentiation, including hyperkeratosis and occasionally intradermal nests (Fig. 2). They were grossly similar to the skin papillomas initiated in utero by dimethylbenzanthracene (Morris et al., 1983). Some of these tumors grew fast and, when they were multiple, killed the hosts. Others showed no sign of progression, while a few regressed at a later age. Few animals which had papillomas during infant life still retained the lesion at the age of 14 months. At 6 weeks of age, corresponding to the peak inci- dence of papillomas, the situation was as reported in Table 111. The Kirsten virus was consistently the most efficient inducer of papillomas with respect to incidence, multiplicity and size. Four out of 142 mice infected on day 8 with KiMSV had papillomas, compared to none in the other groups. This inci- dence increased to 40% following infection on day 10, a significant departure (p < 0.001) from the incidences observed with HaMSV and MoMSV (10% and 15% respectively).

None of the papillomas described in this report became frankly malignant, although in some cases there was wide- spread intradermal infiltration, and a malignant evolution still remains possible for the surviving offspring with papillomas.

Infection at later stages of embryogenesis (from day 11 on) did not produce a single skin papilloma (Table I1 and Rossi et al., 1985). Only sporadic mesenchymal sarcomas were found in C3H and C57BL mice infected during the same periods of gestation (data not shown). It is important to stress here that skin papillomas are rarely, if ever, seen in untreated Swiss mice. However, these animals are highly sensitive to chemical initiation and promotion procedures eventually resulting in squamous-cell papillomas and carcinomas of the skin (Verma

et al . , 1982). To date no viral expression (as revealed by focus- forming assay and reverse transcriptase activity) has been detected in these skin tumors, although we were unable to obtain propagable cell lines from the papilloma explants. In contrast, viral expression by cultured sarcoma cells from the same animals was repeatedly confirmed (data not shown). Lung tumors are a distinctive event associated with the infection of 8-day-old embryos with KiMSV

Tumors (Table 11) were found in the lactating offspring as early as day 15 of age and histologicals were well-differen- tiated alveologenic adenocarcinomas (Fig. 2) affecting one or, rarely, 2 pulmonary lobules and ranging in diameter from 6 to 13 mm. Nine out of 142 (6.3%) animals had this lesion, while an alveologenic adenoma was detected in one newborn treated with KiMSV on day 10 of embryogenesis. These tumors ap- peared to be linked to the biological activity of KiMSV be- cause, although they are spontaneous in old CD-1 mice, they have never been induced with other MSV or with murine leukemia viruses (data not shown and Table 11). Moreover, such large pulmonary masses have not been reported to appear so early in conventional prenatal chemical carcinogenesis stud- ies (Tomatis and Mohr, 1973).

DISCUSSION

Our experiments supplement and extend results of previous studies on the interaction between post-implantation mouse embryos and oncogenic retroviruses, and provide a direct account of the induction of tumors by murine sarcoma viruses in mid-gestation stages. The effects were detected when the infections were performed on days 8 and 10 of embryogenesis, but not before. On day 7 of embryonal growth the various MSVs might have been inactivated by de novo methylation of the integrated retroviral genomes in a way similar to that described by Jahner et al. (1982). Some of these animals, which were infected with KiMSV and then given repeated doses of TPA in the dorsal skin during adult life, had skin papillomas at about 12 months of age as compared to none in the groups receiving TPA or the virus alone (data not shown). The neoplasms caused by KiMSV, HaMSV and MoMSV were generally recognized as originating early in organogenesis by either a marked stage-dependence, displayed for example by lung and skin tumors, or a high cellular polymorphism accom- panying most mesenchymomas and not found in tumors of the same histological pattern induced later in development. In contrast, osteogenic. sarcomas, a group of neoplasms specifi- cally associated with FBRMSV, did not show a similar trend as ascertained by kinetics of growth and morphology. For example, those initiated in mid-gestation embryos were histo- logically indistinguishable from those arising as a consequence of neonatal treatment. Since ossification starts later in preg- nancy, the possibility must be considered that FBRMSV inte-

’I‘UMOKS INDUCED BY MSV IN MOUSE EMBRYOS 265

grates into the precursor osteoblasts without being expressed. endogenous, unknown stimuli are required before the disease Virus expression may occur in more highly differentiated can progress from benign to malignant forms (Hennings et al., cells. 1983).

Our data contradict the long-standing belief that the period Among the retroviruses that we tested, KiMSV W a s by far of development most sensitive to carcinogenesis starts in ad- the most active inducer of skin papillomas, followed by vanced stages oforganogenesis. In addition, infection of mouse €hMSV and MoMSV, while FBRMSV Was ComPleteIY inac- embryos from day 7 of pregnancy onward with the various tive in this respect. There seems to exist a close, albeit unclear, retroviruses that we tested did not result in malformations, relationship between Our data and the reports that chemically with the possible exceptions of brain vascular telangiectasia induced skin Papillomas express the C-Ha-ras oncogene in and of splenic ruptures. These, however, were also observed mice (Balmain et al., 1984; Yamasaki el al., 1987). An under- in neonatally-infected mice. This finding differs from previous standing Of organotropism Of this kind requires characteriza- observations that prenatal chemical and physical carcinogens tion of the stem-celk giving rise to skin Papillomas and a induce developmental abnormalities, but not tumors, when knowledge of how KiMSV, as compared to FBRMSV, disturbs given to pregnant rodents during the major periods of organo- their normal Proliferation. genesis (Tomatis, 1979; Rice, 1981). Oncogene-carrying ret- The detection of alveologenic adenocarcinomas in lactating roviruses may stand apart as a peculiar class of agents offspring infected with KiMSV on day 8 of embryogenesis exhibiting oncogenic but not teratogenic effects in rodents. was another interesting finding of our study. The fact that Such a sharp distinction may be exploited to investigate var- about 6% of infected survivors had such lesions may not be ious aspects of prenatal toxicology such as that of genomic fortuitous, since roughly the same incidence of lung tumors is components possibly leading to abnormal development and to expected to occur in untreated CD-1 mice kept alive for their cancer, as well as the mechanisms by which MSV may affect entire lifespan. The greatly reduced latency and time-depen- cellular regulatory and structural functions likely to result in dence exhibited by these tumors, together with our results on the initiation and maintenance of developmental tumors. the occurrence of skin papillomas, gives an intriguing example

The appearance of skin papillomas following MSV was the of the possible role played by oncogenic retroviruses in pre- most important single finding of our study. The period of natal carcinogenesis. The most relevant observation is, per- sensitivity started around the onset of organogenesis (about 15 haps, that tumors, which are spontaneous to or inducible only somites), was maximal on day 10 of embryogenesis (about 35 in adult CD-1 mice, may also be induced at mid-somite stages somites) and abruptly fell to zero from day 11 of pregnancy by KiMSV; this represents a means of testing the hypothesis onward. Such tumors have not been previously induced in the that tumors may fmpently arise in target embryonal cells. newborn by means of prenatal exposure to carcinogens before pjaccntation. However, transplacental chemical initiation and post-natal promotion of skin tumors have been reported in mice (Goerttler et al., 1980; Napalkov ef al., 1987). In addi- tion, cutaneous squamous tumors have been observed in trans- genic mice carrying the bovine papillomavirus genome and in mice transfected with v-Hams during adult life and then treated with tumor-promoters (Lacey el al., 1986; Brown et a/. , 1986). The fact that in our study none of the animals had squainous carcinomas suggests that additional, exogenous or

ACKNOWLEDGEMENTS

We thank Dr. L. Tomatis for helpful comments on the manuscript, Dr. R. Puntoni for statistical analysis of the re- sults, Mr. G. Campanile and Mr. M. Cipolla for histological preparations, Mrs. T. Barone and Mrs. M. Isola for editorial and photographic assistance and Ms. S. Beltramea for typing. This study was supported by grants from the CNR “Progetto Finalizzato Oncologia”.

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