induction in hamsters of various carcinomas and sarcomas by in

Induction in Hamsters of Various Carcinomas andSarcomas by In-Vitro SV40-Transformed HomologousEmbryonic Skin and Subcutaneous Tissue Cells

Role of Target Ceals in Determining Tumor Morphology

George Th. Diamandopoulos, MD,* and Mary F. Dalton-Tucker, BA

TME caCoSCOPWc MORn OLOG FE&TURES of various malig-nant neopl in the human are utilized quite often in attempting toascertain p or in deciding on the proper type of therapy. Never-theless, apart from this utilitarian aspect of tumor morphology from amore basic point of view, the undertanding of the factors that deter-mine tumor morphogenesis is of great biologic interest Thus, atptshave been made to determine if the morphogenic potential resides with-in the inducing etiologic factor or if it is a reflection of the genotype ofthe neoplastically transformable target celL

Since it ahleady has been established that in DNA- i enesisthe viral genome plays a pminent role in the induction of the oncogenicstate, vanous investigators,' including ourelves," have endeavored todiscover its role in determining tumor morphology. Suffice it to say thatas of now no agreement has been reached > as to the relative impor-tance of the viral genome in influencng morphogenesis. The answermay lie in the fact that different meanims may be operational fordivergent virus-ll systems 8 and, therefore, extrapolations from onesystem to the next are not warranted On the basis of this interpretationwe have attempted to answer the question of tumor morphogenesis as itconcerns the simian virus 40 (SV40)-hamster cell system only. For ex-periments presented in this communication we have utilized hamstrembryonic skdn and subcutaneous tissue cells, transformed neoplasticallyin vitro under the influence of the oncogenic agent SV40.

From the Deartnumt of Pathology, Harvard Medical Sdcoot Boston, Mass.Suppurted by US Public Health Service Reari Grant CA0731, fro the Nathonal

Canc NIH B a, Md.ped for publition Mardc 24, 19w9.

Addres for reprint requsts: Dr. George Th. nd , Department of Pathol-ogy, Harvard Medical Sdcool, 25 Shattuc St, BostDn, Mass 02115.

DRecpiet of US PublicH Service Erch Career Development Award 1-K4CA 13,444-01 fm the Natonal Istitute, NIH

59

60 DIAMANDOPOULOS AND DALTON-TUCKER

Materials and Methods

Virus

SV40, Strain VA 45-54, was propagated in grivet monkey kidney cells, LineAH-1.14 The infectivity titer of the stock virus was 106.5 median tissue cultureinfective dose (TCID,0) per 0.1 ml, as determined in the AH-1 cell system.

Cell Cultures of Hamster Embryonic Skin and Subcutaneous Tissue

Two female golden Syrian hamsters (Cricetus auratus), when approximately13 and 15 days' pregnant, were obtained from a local supplier. The 11 embryosof the first pregnant hamster and the 9 more-mature embryos of the second onewere removed aseptically. The two litters were kept separate and were labeledI and II, respectively. Extensive areas of the skin and subcutaneous tissue of eachembryo were removed surgically, pooled with similar material from embryos ofthe same litter, then minced and trypsinized. Cell suspensions containing roughly5 X 105 cells per 1 ml of growth medium (basal medium Eagle with 10% fetalcalf serum and proper amounts of antibiotics-penicillin 50 units/ml, streptomycin50 ,ug/ml, and amphotericin B 1 jg/ml), were prepared separately from embryosin Litters I and II. Half of the cells from each suspension were exposed to un-diluted stock virus (0.1 ml virus for every 1 ml cell suspension), and the re-mainder were kept as controls. Both SV40-exposed and nonexposed cells werethen grown in a stationary position at 370 C as monolayers in 16 X 150 mmtubes (1 ml of cell suspension per tube), as well as in 4- and 12-oz soft glassbottles, containing 10 and 25 ml of the cell suspension, respectively.

Sexial cell passages from the virus-inoculated and uninoculated primary cellcultures of both Litters I and II were initiated at the end of Weeks 2, 4, 8, and12 following the setting up of the experiment. Subsequent passages of oell culturesleading to the eventual establishment of penranent cell lines were made everyweek or every other week depending upon their ability to form a monolayer. At eachpassage, the cultured cells were divided in a ratio of 1:2. With the initiation ofsexial cell passages, the type of growth medium used was Puck's 10-10. Thisconsisted of Puck's N-16 medium containing 10% fetal calf serum, 10% heat-inactivated horse serum, 4% NCTC-109, 0.5% lactalbumin hydrolysate (Earle's),and proper amounts of antibiotics (penicillin 50 units/ml, streptomycin 50 ,gg/ml,and amphotericin B 1 jg/ml). At the end of Days 1, 3, 5, and at Weeks 2, 4,8. and 12 following initiation of the experiment, tube cultures from the virus-inoculated and uninoculated cells of both Litters I and II were fixed and stained,employing the collodion-embedding technique."' A tube culture of each of theastablished cell lines from the passage preceding cell implantation into animalsalso was fixed and stained.

Storage of Cells

Cells from representative passages of all SV40-exposed and of the five survivingnonexposed lines were suspended in "freezing medium" 6 in concentrations varyingfrom 2 X 106 to 3 X 106 cells per milliliter. Aliquots of 1 ml of cell suspensionwere placed in Pyrex glass tubes (13 X 100 mm) and were heat-sealed. Thespecimens were frozen slowly and stored at -700 C. When warranted, cetainampules were quickly thawed at 370 C and the cells were again grown in vitro.

Vod. 56, No. I

SV40 AND TUMORS 61

Immunofl1uoce TechniqueThe inir t test for the ianuclear SV40-inuced T (tumior) antigen was

em.pkyed1 on six SV4-exposed es and one line seleted at ndomfro both Litters I and IL

Te for OncoenicityAt 1-3 follwing initiatim of suculivation (Table 1), cell susp Ms

of all the SV40-epsed and of the five s i cell lines wereprqered. Aliquots of 0.5 ml containing axiately 5 X 10 cells, were im-plantedsubpthelially into both cheek of weanling (21-day-old) maleSyrian hamste, ading top e ly descibed For the cheekpouch rou of implantioa , 3 animals wee utlzed for each cell lin.1 Moreover,5 X 106 cs of each line, nad in 05 ml of gowth meinu, wee iplantedbliaterally in 2 additional weanling hamstm, at subcutaneous and at intauscularsites At monthly intervals the anima were anestheized, and the dcek pouch, and

san and tra sular ss of cell implantation were ai At theend of Months 1, 2, and 3 folling cell implantation, representative animalsbearing tumors were and a comp necropsy was performed. The tumorsand viscm were fimed in Bouins and then sectoned for micmocopievaluatoL AR animals, whether or not they were bearing tumors, were sacificedat the end of 3 months of observaton.

ObservahomnTramation In Vlb Differ e B n Conrl andSWlnwh CuwPrimary Embryonic Sldn and a Tssue Cell CulturesA tDtal of 100 such cltures were ncluded initially: 50 were fo Litter

I and the other 50 fr Litter II. Half of the cell culures bothTable 1. History and Morpholic Pr ti of 34 Oncogenic SV40-TransfofnedHamtr Embryonic Skdn and Subcutanos Tssue Cell Unes

Initition of Animalsubcultlv.tko Inocullation No. of lines No. of tumors

Lkns (k)* (wk) Carcinom/Sarrcomes Carcinomas/Sarcom

Utter IA-E 2* 12* 2/5 6/31F-J 4 14 1/5 1/29K-0 8 16 0/5 0/15P-Q 12 18 0/2 0/9

LUter IIR-U 2 7-10 0/4 0/21V-Y 4 10-12 1/4 3/13Z-2C 8 12-16 0/4 0/14

2D-2H 12 16-18 0/5 0/20

SuboasLtter IA-Q 2-12 12-18 3/17 7/84

UtLlr IIR-2H 2-12 7-18 1/17 3/68

TotalsLtter I & IIA-2H 2-12 7-18 4/34 10/152

Intwrvals in a_l following SV40 expur of prmawry cl cultures.

July 1969


litters were inoculated with SV40 and the remaining halof each servedas uninoculated controls.

Examination of fresh, as well as of fixed and stained, primary cellcultures revealed slight to moderate increase in cell populations in theinoculated cultures as compared to changes in the controls. This featurewas also reflected by differences in the number of mitoses in the twogroups of cell cultures. Moreover, as early as 3 days following SV40exposure, polynucleate giant cells were seen. Their identification wasrevealed more easily in the stained preparations. The number of thesecells gradually increased, so that by the end of Week 2 they were morenumerous and occurred in clusters. Concomitant with the increase inthe number of giant cells, an overall cellular pleomorphism began to ap-pear, which progressed in complexity with the passage of time. Gradu-ally, this pleomorphism was accompanied by focal heaping up of cells,advancing to a more diffuse cellular multilayering. Similar cell changesof severe pleomorphism and extensive multilayering were not seen inthe control cultures. Only an occasional tube culture showed a rarepolynucleate giant cell or a discrete focus of well organized multilayer-ing.

Attempts were made to group all primary cell cultures according towhether or not they contained epithelioid cells. All such efforts were invain, since it was shown repeatedly that foci earmarked as consisting ofepithelioid cells became more pleomorphic with the passage of time, andpleomorphic cells gradually developed features resembling those ofepithelial cells. Our inability to identify epithelioid elements in primarycell cultures was more apparent in the virus-inoculated than in the con-trol cell cultures. This was to be expected, since the morphologic char-acteristics of the nonvinus-exposed cell cultures were relatively stable atvarious time intervals.

In addition to the aforementioned morphologic differences elicitedbetween the virus-exposed and nonexposed primary cell cultures, differ-ences in metabolism were identified. As early as 1 week following initia-tion of the experiment, there was consistent difference in the pH of thegrowth medium-the SV40-inoculated cell cultures acidified the mediummore rapidly and more extensively than did the controls. This differencein the two groups of cultures persisted and became more pronouncedwith the passage of time. Similar differences in metabolism betweenSV40-exposed and nonexposed cultures have been described by variousinvestigators 18 in other oncogenic virus-cell systems as well.

Vol. 56, No. I

SV40 AND TUMORS 63

Subcultures

As stated previously, at regular time intervals a senes of subcultureswas initiated fr the primary embryonic skin and subcutaneous tissuecell cultures. A total of 17 lines frm the virus-inoculated and 17 fromthe control cultures from Litters I and II were begun. Of the 34 controlcell cultures, only 5 survived beyond four to five subcultivations, produc-ing adequate numbers of cells for animal inoculation. The cells of theremaining 29 control cultures failed to multiply beyond the third orfourth passage, although subcultivations were attempted only at intervalsof 2-3 weeks. On the contrary, all 34 SV4 0eped cell cultures fromLitters I and II could be subcultured routinely every week, giving rise toestablished cell lnes. In fact, if the subcultivation was attempted everyother week, there was ov h of cells with their subsequent detach-ment from the glass, and death. Portions of each of the 34 cell lnes werefrozen and stored at -700 C. Three of these lines were thawed andwere again propagated successfully in vitro through additional passages.Their in-vivo oncogenicity also was tested.Fr the firt passage on, the celular pleomorphism that was seen in

all 34 SV40-exposed primary cel cultures became progressively morepronounced. With continued subcultivations the cytologic features werecharactertic of anges previously described as in-vitro SV40-mediatedmorphologic tbnsformation.1-4 It consistd of (Fig 1-4) profoundcelul pleomorphism, with the appearance, in some cases, of numerouspolynucleate giant cells. ITere was also nuclear and cytoplasmic hyper-chroinasia, and an increase in the number of mitotic figures, many ofwhich were abnonnaL As was the case with the primary cell cultures,repeated attempts failed to any one of these establishedSV40-transformed cell lines as being predominantly epithelioid. How-ever, in some of these lines foci of epithelioid cells were identified attimes. Their persistence, however, could not be followed through sub-sequent passages. Even when stained preparations from each of theselines were examined retrospetively-ie, with the knowledge of thehistologic type of tumor they had induced, no in-vitro morphologic fea-tures could be elicted which were pathognomonic of an epithelial poten-tial in vivo.The five control cell lines that survived at least four to five subcultiva-

tions, at which time they were tested for oncogenicity in the appropriatehost, showed slight pleomorphism, absence of hyperchromasia, andminimal numbers of mitotic figures, all of which appeared normaL Thisprofoundly dimini growth potential of all five control lines also was

July 196


revealed by their metabolism, as reflected in their inability to acidify thegrowth medium to the extent and speed of that found in any of the 34SV40-transformed lines. Attempts to identify epitheioid cells in the fivecontrol lines were also in vain. No retrospective evaluation of the homo-logous, stained preparations could be carried out, since these lines werenot oncogenic, as will be seen subsequently.Evidence that the viral genome was present in the SV40-transformed

cells was obtained by the application of the indirect immunofluorescencetechnique for the intranuclear SV40-induced T antigen, in six SV40-ex-posed lines selected at random-four from Litter I (Lines A, F, J, andQ) and two from Litter II (Lines 2G and 2H). This antigen was demon-strated in 100%o of the cells of all six lines. It was not found in the onecontrol line (Line 2E') from Litter II, which had also been selected atrandom.

Oncogenicity In Vivo

Cells of the five surviving control and the 34 SV40-transformed lineswere tested for oncogenicity in the homologous host. Moreover, thethree previously frozen and subsequently thawed transformed cell linesalso were tested for oncogenicity. Although results regarding the trans-formed lines are summarized in Table 1, data from the control lines arenot included because they were negative-ie, none of the five controllines induced tumors in cheek pouches or in other sites of cell implanta-tion of any animal in which it was tested. Microscopic examination ofsome of the cheek pouches inoculated with cells from these lines revealedno neoplastic cells. An occasional pouch, however, showed minimalfibrosis and infiltration by small numbers of mononuclear cells.

In contrast to the above negative findings regarding the five controllines, cells of all 34 SV40-transformed lines induced tumors in the cheekpouches, as well as in intramuscular and/or subcutaneous sites of theinoculated animals. Although the great majority of the lines producedtumors in all three sites of implantation, occasionally a given line pro-duced a tumor in only one of the two cheek pouches of the same animal,while another line might produce tumors at the cheek pouches andsubcutaneous sites, but not intramuscularly in the various animalsutilized.The microscopic appearance of the tumors arising from transformed

cell lines of Litters I and II will be discussed separately (Table 1). Of the17 lines of Litter I, 3 (Lines A, B, and F) gave rise to both epidermoidcarcinomas and sarcomas (Fig 5-7), while the remaining 14 induced ahistologic variety of sarcomas only, from the well differentiated spindle-

Vol. 56, No. 1

SV40 AND TUMORS 65

cell type (Fig 9) to the pleomorphic type with few or many polynucleategiant cells (Fig 10). The epidermoid carcinomas that arose from thesethree lines were not in any way "pure." They were always surroundedby, or ingrown by, sarcomatous elements. Moreover, in some of thesetumors only small clusters of carcinomatous cells could be identified(Fig 5), located in the midst of sarcomatous neoplasm. However, inthose tumors in which large areas of well-differentiated epidermoidcarcinoma were seen, the squamous cells in the central part of the tumorappeared to produce keratin, and in some cases even formed "epithelialpearls" (Fig 6). No keratin formation was found in the smaller foci ofepidermoid carcinoma (Fig 7), although the cells of which they werecomposed appeared to be relatively well-differentiated epithelial ele-ments. Aliquots of cells of these three carcinoma-inducing lines, whichhad been frozen and stored previously, were thawed and again tested foroncogenicity in vivo. Although all three lines were shown to be onco-genic, only one (Line B) gave rise to epidermoid carcinomas in two offive tumors examined. The other two lines (A and F) produced sarcomasonly. The only other feature worthy of note was the microscopic ap-pearance of one of the tumors produced by Line F which, althoughsarcomatous, consisted of cells forming a rostte-like pattern. This mor-phologic type of sarcoma usually has been associated with tumors in-duced experimentally by human adenoviruses only, particularly theadenovirus type 12 This point will be elaborated upon in the discussion.

All 17 SV40-transformed lines of Litter H gave rise to the wholehistologic spectrum of sarcomas, from the well- to the poorly-differ-entiated varieties. Only one of these lines (V) produced adenocarci-nomas (Fig 8) in addition to sarcomas. Proof that the tubular (adenoma-tous) elements had been induced by the transplanted neoplastic cells,rather than representing host normal glandular strctures, was the factthat they were found in tumors of three different animals-two in thecheek pouch, and one at an intramuscular (Fig 8) site. Finally, one otherline (W) produced a sarcomatous tumor, the cells of which formedrosettes (Fig 11 and 12), which were histologically similar to neoplasms(as mentioned above) produced by human adenovirus type 12

All tumors described above were malignant not only morphologically,but in their behavior as well (Fig 13-16). They invaded locally, de-stroying surrounding normal structures. Many of them metastasized todistant organs. The majority of animals showing metastases were pri-marily those bearing tumors for 2-3 months at intramuscular sites of cellinoculation. Quite often, however, cheek pouch tumors metastasizedalso. The lungs (Fig 15) were the organs most often involved, but the

July ig


kddneys (Fig 16), adrenals, and heart occasionally exhibited metastaticfoci.As previously reported,L7 clusters of mononuclear cells (Fig 13) con-

sisting of plasma cells and lymphocytes were seen within and aroundthe periphery (particularly perivascularly) of many of the tumors ex-amined histologically, as well as, but to a lesser extent, in the vicinity oftumor metastases in distant organs. Although lytic changes of the neo-plastic cells were seen at times in the vicinity of the mononuclear ele-ments, their causal relationship could not be established on the basis ofthe material under study.

DiscussionFrom the data presented in this communication, it appears that ham-

ster embryonic epidermal cells and/or their precursors can survive, sub-ject to in-vitro cultural conditions. Moreover, it is evident that such cellsare capable of transforming neoplastically under the influence of theoncogenic virus SV40, whereupon they give rise to epidermoid carcino-mas when implanted in the homologous host. It also has been shown thatother cutaneous epithelial cells of glandular potential can survive andtransform by SV40 in vitro, and then give rise to adenocarcinomas invivo.Although the phenomenon of in-vitro survival and neoplastic trans-

formation of epidermal and dermal epithelial cells has thus been es-tablished, it is evident that such an event takes place rarely or, at best,occasionally. This is attested to by the fact that of the 34 SV40-trans-formed embryonic sldn and subcutaneous tissue cell lines studied, only4 gave rise to carcinomas, while the remaining 30 induced a wide his-tologic variety of sarcomas only. Thus, our findings give supportiveevidence to the widely held view that fibroblastic and other mesen-chymal elements survive under cultural conditions much easier thantheir epithelial counterparts, and are more readily transformed neo-plastically by various oncogenic viral agents.The nature of the factor(s) responsible for these profound dif-

ferences between epithelial and mesenchymal elements in regard to theirin-vitro growth potential or to their susceptibility for virus-mediatedneoplastic transformation is not known at present. In this respect, how-ever, two fidings emerge from an analysis of the present data. First, nocarcinomas were induced by any of the SV40-transformed lines whichwere initially subcultivated later than 4 weeks following virus exposure,or were implanted into the appropriate host later than at 14 weeks(Table 1). This may imply that most, if not all, of the epithelial elements

Vol. 56, No. 1

SV4O AND TUMORS 67

or their s died relatively early, before (or even after) virustransformation, either in vitro or in vivo, while on the contrary, themesenchymal elements survived and eventually established lnes ofsarcomatous potential Further proof that selective pressures exist whichfavor survival of mesenchymal over epithelial elements is shown by ourfinding that carcinoma-producing cells were unable to endure freezingand storage in two of three lines tested. Second, the three SV40-trans-formed lines that induced all the epidermoid carcinomas belonged toLitter L and noe to Litter II. As is well remembered, embryos of LitterI were less mature tn tiose of Litter II. It may be possible that well-differentiated epidermal cells or their s have a better ance tosurvive in vitro or to transform oncogenicaly if they are derived fromless mate embryos. Experiments are now in progess in an attempt toclrify isupposition.Another point the prsent data emphsize is that in the SV40-hanster

cell system the genetic makeup of the transformable target cell deter-mines tumor morphology: It is quite apparent that no histologic featuresof the tumors are found that are ca istc of an SV40-mediatedneoplastic ange. On the contrary, both carcinomas (epidermoid oradeno-) and sarcomas may be induced This implies that the appropriateepithelial or mesenchymal cell is the one that de morphogenesisof the neoplasms. If it were to be shown conclusively that the SV40 ge-nome tuly lacks any (neoplastic) morphogenic pntial, it would notfollow that this should be the case with all known oncogenic viruses. Itis quite possible, and perhaps very likely, that different mecbani maybe operational in viruses which vary so widely among themselves, notonly in terms of their nucleic acid content (RNA versus DNA), but par-ticularly in the number or kind of genes partaking in the induction ofthe neoplastic process. On the other hand, it is still possible that whathas been designated by soMe investigators >1- as virus-mediated mor-phogenic potential may be the result of increased susceptbility of somecells over others in regard to infection and sformation by a givenvirs. It has been thught in the past 1- that experimentally virus-induced sarcomas which exhibit a rosette-lke pattern are pathognomonicof hmnnanad type 12-mediated neoplastic transformation. Never-theless, our present data show that histologically similar tumors havinga roette-like pat, which is suggestive of neuroectodermal origIn,S8may be produced when the appropriate Hells-ie, embryonic skin andsubcutaneous tissue-are exposed to SV40. However, the very smaIlnumber of these neoplasms detected in the present experments (2 of152 tumrs emined histologically), should make us very cautious of

July 196


our extrapolations. Additional data are needed therefore in order toclarify this point.A brief comment should be made in regard to another of our observa-

tions, since we have elaborated upon it in the past rather extensively.'7We again have noted a lymphocytic and plasma cell reaction aroundand within the majority of these experimentally induced tumors, both atthe primary sites of tumor growth and in many of their metastases. Thecomposition of the cellular infiltrate and the evidence for degenerativechanges seen in some of the SV40 tumor cells located in the vicinity ofthe mononuclear reaction is again interpreted as representing a hostdefense mechanism to the tumor, probably of immunologic character.As previously stated,8 the extent and magnitude of this mononuclear cellinfiltrate could not be correlated with the overall tumor aggresgivenessor lack thereof. Further experiments are still in progress.

SummaryIt has been found that when cells derived from Syrian hamster em-

bryonic slin and subcutaneous tissue are transforned neoplastically invitro by the oncogenic simian virus 40 (SV40), they develop cytomor-phologic characteristics divergent from those of the primary nonvirus-exposed cells. However, no cultural features of the transformed cellsare invariably identifiable that can be utlized in predicting the histologyof the neoplasms they will induce when implanted into the homologoushost.

Although the majority of these neoplasms represent a morphologicvariety of sarcomas, there is a small proportion that also contain well-differentiated epidermoid carcmomas, some of which produce keratin.Moreover, a rare tumor contains foci of adenocarcinoma. From thesedata, it seems that the keratinogenic or potentially keratinogenic em-bryonic epidermal cells are the progenitors of the epidermoid carcino-mas, and that the glandular dermal elements are the progenitors of theadenocarcinomas. Furthermore, it was found that among the neoplasmsdiagnosed as sarcomas, a rare one consists of cells- forming a rosette-likepattern, which is strongly suggestive of neuroectodennal origin. Thismorphologic feature had been considered in the past to be found inexperimental tumors induced by human adenoviruses only, particularlythe adenovirus type 12.The conclusion, therefore, may be drawn that in the SV40-hamster

cell system there is lack of any significant virus-mediated morphogenicpotential. Instead, the cytomorphology and histopathology of the neo-plasms are determined primarily by the genotype of the virus-transform-

Vol. 56, No. 1

July 199 SV4 AND TUMORS Q9

able target ells that in th Factors tD a mt for these phe-it-ne are

RefBieeWne1. ENIS, J. F. Cell tanIFormatwi by viruses as ilustrated by the response

of human and hanster renc tD vir 40. Harey Lect 59:113-153, 1965.

2. RAMON, A. S., and r , R. LnuLiOf malignancy in virin new rn hamstr kidney tissue inf with mian c ting(SV40). Proc Soc Exp Bkil Med 111:323-28, 1962.

3. BL.Acr, P. H., Rowa, W. P., and CooEtx, H. L An analysis of SV40-ndixedtrnsformation of mster kidney tissue in vitro. IL Studies of three cloedIrived frmm a cexmtnucus line Of transform celk Proc Nat Acad SA USA50:847-85, 1963.

4L Sr, H. M. Nopla trsrm ation i by simin viru 40 in Syriamhamst n ogial and menxmgeal cell utures. Arch Ges Virrwh22:122-142, 1967.

5. DzmmD V., and ImAN, J. M. Transformation of hamstw embryoclsin o by polyan virus: Morphologicat karyological, immnlogl and

antntion cdmC t- . I CeUl Physiol 66:351-409, 1965.6. DIAwM oo CosC. TEL, and EN 7, J. F. Comparison of the cylmor-

phologic charistcs of vitro SV40-rsformed hamst embryo cEwihi the histologic featmes of the neoplasms which they induce in themx~logus bhost. Ame I Path 49:397-417, 1966.

7. I oAPO , C. Tn Comparison of the ir cytogy of hasreanhy cell I trnsormed spontaneously" or by SV40: wi contastof sarxxnasinduced in the homolgu host. Ame I Path 52:63347,1968.

8. DGIA wOPOULOS, C. TEL Histo hology of sariLas iduc hastesby clnes of in-vitro SV40-trasfo homol s heart cells. Attemptsto erg fetue and degree of cpotil MerI Path 53:753-767, 1968.

9. SrANNEas, C. P., Tna, Jo E., and SnwNovrrcK, L Studiesonthe htranForma-tkin of ams b cels in cuue by polyna viu. L Propeqries oftransformed and normal cel. Virlogy 21:448-43, 1963.

10. TonA, G, J., HAB=L, K., and Gmw, H. Anti ic and c l propatiesof cells doubly trnFo by polywma virus and SV40. Virology 27:179-185, 1965.

11. MnmA, D., and SAcKs, L A caunse of variation in clonal morphology ofpolyoma t nsrmed hamster cells. Vfrology 27:39-408, 1965.

12. Tmzmo-ro, K. K., and HABEL, K. Hamst tumor cls doubly t nsfomdby SV40 and polyoma viruses. Virology 30:20-28, 1966.

13. Waxs, S. A., JR., RAsNO, A. S., MALicRN, R. A., and KrzcEr$, A. S.In vitro neopasfic asformation of newbn hamster salhvary gland tissueby o DNA viruses. Cancer 19:1411-1415, 1966.

14. GCt)r.P, A. Grwth and cypaic ect of rubella vrs in a line of gremmonkey kidney cells Proc Soc ETp Biol Med 118:85-90, 1965.

15. ENaus, J. F., and Pm.EEs, T. C. Propagation iin tissue cultures of cyto-

70 DIAMANDOPOULOS AND DALTON-TUCKER Vol. 56, No. 1

pathogenic agents from patients with measles. Proc Soc Exp Biol N.Y. 86:277-286, 1954.

16. DxAwmAoPouLos, G. TH., and ENDERS, J. F. Studies on transformation ofSyria hamster cells by simian virus 40 (SV40): acquisition of oncogenicityby virus-exposed cells apparently unassociated with the viral genome. ProcNat Acad Sci USA 54:1092-1099, 1965.

17. DImAmjNoPouLos, G. TH. Plasma cell and lymphocytic reaction in theSyrian hamster to transplanted homologous tumor cells transformed in vitro"spontaneously" or by SV40. Arch Ges Virusforsch 22:108-121, 1967.

18. OGAWA, K., TsuTsUMI, A., IWATA, K., FujI, Y., OHmoRI, M., TAGUCHI, K.,and YABE, Y. Histogenesis of malignant neoplasm induced by adenovustype 12. Gann 57:43-52, 1966.

19. Spjur, H. J., VAN HOOSIER, G. L., and TRENTIN, J. J. Neoplasms in ham-sters induced by adenovirus type 12. Arch Path (Chicago), 83:199-203,1967.

20. NEIDERS, M. E., WEISS, L., and YOmN, D. S. A morphologic comparison oftumors produced by type 12 adenovirus and by the HA-12-1T line ofadeno-12 tumor cells. Cancer Res 28:577-584, 1966.

21. VAN DER NooRDA, J. Transformation of rat kidney cells by adenovirus type12. J Gen Virol 2:269-272, 1968.

The SV40, Strain VA 45-54, was obtained originally from Dr. M. R Hillan ofthe Merck Institute for Therapeutic Research, West Point, Pa.

Legends for FiguresIllustrations were prepared from cell cultures and tissue sections that had beenstained with hematoxylin and eosin.

Fig 1-4. Cytologic morphology of cell cultures of four lines of in-vitro SV40-trans-formed hamster skin and subcutaneous tissue cells. X 800.

Fig 1. Line A, passage 6. Although the line appears slightly pleomorphic, it inducedepidermoid carcinomas (Fig 5), as well as sarcomas.

Fig 2. Line 0, passage 5. Although the line appears epithelioid, it induced sarcomasonly.Fig 3. Line P, passage 3. Although the cells appear stellate, this line induced thewhole histologic spectrum of sarcomas, from the spindle-cell type (Fig 9) to themore anaplastic type with many polynucleate tumor giant cells (Fig 10).Flg 4. Line R, passage 3. This line exhibits profound pleomorphism with many giantcells. It induced a variety of sarcomas.

SV40 AND TUMORS

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July 1969 71

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Fig 5-8. Sections of carcinomas arising in hamsters at various sites of inoculationfrom four lines of homologous in-vitro SV40-transformed skin and subcutaneous tissuecells. x 800.

Fig 5. Epidermoid carcinoma, surrounded by spindle-cell sarcoma. Line A, passage 6.Injection site: cheek pouch.

Fig 6. Well-differentiated epidermoid carcinoma with central keratinous nests ("epi-thelial pearls") surrounded by undifferentiated sarcoma. Line B, passage 6. Injectionsite: cheek pouch.

Fig 7. Epidermoid carcinoma in close proximity to poorly differentiated sarcoma. LineF, passage 5. Injection site: cheek pouch.

Fig 8. Well-differentiated adenocarcinoma in the midst of sarcoma. Line V, passage 3.Injection site: intramuscular.

Vol. 56, No. 1

July 1969 SV40OANDTUMORS 73

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Fig 9-12. Sections of sarcomas arising in hamsters at various sites of inoculationfrom two lines of homologous in-vitro SV40-transformed skin and subcutaneous tissuecells. x 800.

Fig 9 and 10. Spindle-cell sarcoma, and pleomorphic sarcoma with many polynu-cleate tumor giant cells, respectively, representing two areas in the same section of atumor. Line P, passage 3. Injection site: cheek pouch.

Fig 11 and 12. Sarcoma exhibiting a rosette-like pattern, from two areas in the samesection of a tumor. (Note: This type of sarcoma usually has been associated withtumors induced experimentally by human adenoviruses only, particularly adenovirustype 12). Line W, passage 3. Injection site: cheek pouch.

Vol. 56, No. 1

SV40 AND TUMORS 75

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Fig 13-16. Intravascular tumor emboli. x 800.

Fig 13. Intralymphatic tumor embolus at periphery of a sarcoma. Note mononuclearcell infiltration at periphery of the tumor. Line T, passage 5. Injection site: intramuscular.

Fig 14. Intracapillary or intravenular tumor embolus at periphery of a sarcoma. LineH, passage 5. Injection site: intramuscular.

Fig 15. Intravascular tumor embolus in the lung. Line D, passage 6. Injection site:cheek pouch.

Fig 16. Intra-arterial tumor embolus with infarction of the renal parenchyma. Line H,passage 5. Injection site: intramuscular.

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