Int. J . Cancer: 9, 57-65 (1972)

FELINE FIBROSARCOMA VIRUS: QUANTITATIVE FOCUS ASSAY, FOCUS MORPHOLOGY

AND EVIDENCE FOR A " HELPER VIRUS "

Ruth MCDONALD Lauren G. WOLFE and Friedrich DEINHARDT Department of Microbiology, Rush-Presbyterian-St. Luke's Medical Center,

1753 West Congress Parkway, Chicago, Illinois 60612; and Graduate College, University of Illinois at the Medical Center, Chicago, Illinois 60612, USA

Feline fibrosarcoma virus (FeSV) was assayed by focus formation in feline and marmoset fibroblast cultures overlaid with agar and re-fed with fluid media. The FeSV titers were consistent from experiment to experiment and were about four times higher in feline fibroblasts than in marmoset fibroblasts in parallel assays, Clmes of FeSV- infected cells grown in soft agar, and foci of transformed cells produced by FeSV in monolayer cell cultures were both of two morphological types: round cells (r) and a loose meshwork of fusiform and round cells (fr). Thermal stability experiments indicated that the morphological differences of the foci were virus-dependent.

The titration pattern of FeSVgrown in marmoset or feline cells indicated that these preparations were either competent or contained an excess of helper virus. Preliminary challenge experiments with Snyder- Theilen FeS V grown in marmoset cells indicated that a 10- to 100-fold excess of non-transforming, interfering virus (helper virus) was present in this FeSVpreparation.

Several strains of C-type RNA viruses have been isolated from naturally occurring feline fibrosarcomas. Two of these, the Snyder-Theilen strain (ST-FeSV) and the Gardner strain (G- FeSV) (Snyder and Theilen, 1969; Gardner et al., 1970) induced fibrosarcomas in white- lipped marmoset monkeys, Saguinus fuscicollis, S. nigricollis (Deinhardt et al., 1970), and trans- formed marmoset and feline cells in vitro (Wolfe et al., 1970). Although the transformation of human, canine, and feline fibroblast cultures by G-FeSV and ST-FeSV without an agar overlay, and the use of such cultures for focus assays, were reported recently (Sarma et al., 1970; Sarma 1971; Sarma and Log, 1971) assays of FeSV in cell cultures with only fluid overlay gave inconsistent results in our laboratory. We attempted therefore to obtain focus formation of

FeSV-infected cells under an overlay which would minimize dissemination of transformed cells or the spread of virus. Various modifications of existing focus assay techniques were evaluated for their ability to support optimal expression of cell transformation.

MATERIAL AND METHODS

Virus

ST-FeSV and G-FeSV were supplied by Dr. Theilen, University of California, Davis, Cali- fornia, USA, and Dr. Rickard, New York State Veterinary College, Ithaca, New York, USA. Stocks of ST-FeSV and G-FeSV were prepared from cat tumors by the Moloney technique (Moloney, 1960) and stored in aliquots at -120" C. ST-FeSV was also prepared from a

Received : September 20, 197 1. Postdoctoral Fellow, National Research Council of Canada.

57

MCDONALD ET AL.

tumor-cell line derived from a ST-FeSV induced sarcoma in a marmoset (CO-1). The medium was removed from the CO-1 cell cultures and was kept at 0-4" C while the cell sheets were frozen and thawed three times. The medium was then re- combined with the disrupted cells and centri- fuged at 1 ,500xg for 30 min. Thz supernatant, designated CO-1-ST-FeSV, was stored in por- tions at -120" C and was filtered through a 0.45 p filter before use. Four harvests of the CO-1-ST-FeSV were made; the first from the 11th passage of the CO-1 cell line, the second from the 13th passage, the third from the 39th and 40th passages, and the fourth from the 62nd passage.

Cell cultures

The type and origin of cell cultures are given in Table I . Cell cultures were grown in plastic flasks with standard techniques and Eagle's improved

basal medium in Hank's balanced salt solution (BMEI-H) supplemented with 10-15% unheated fetal calf serum (FCS), 0.19% sodium bicar- bonate, and 2 mM glutamhe, 100 units penicillin and 50 pg streptomycin/ml. With the exception of two secondary marmoset kidney cultures, all cultures were prepared from cell lines between passages 2 t o 30 after explantation.

Focus assay

The final standard technique and some of the variations evaluatcd in this study are listed in Table 11. In the standard technique the indicator cells were seeded in 30 ml Falcon plastic T- flasks in 5 ml of BMEI-H supplemented as described under cell cultures. The cells were incubated at 37" C, and 1-18 h later, when the cultures had formed a n approximately 60% confluent monolayer, the culture medium was removed and 0.2 ml of 2-, 5- or 10-fold dilutions

TABLE I

CELLS SUSCEPTIBLE TO CO-I-ST-FeSV IN NON-OVERLAID CELL CULTURES

Origin Designation Type of culture

0 r g a n i s m Tissue Source

Human Embryonic

Marmoset Kidney lung

Skin

Muscle Embryo Cat

Tongue

Muscle

Wi-38

Secondary cultures

TV-106 MF-69CB2S

MF-69CBI M FEF-R

FEF-F.L.9202 (Code No. FFc 9 WF)

(Code No. FFc 71 WF)

(Code No. FFc 3 Tg) TE 7050

FEF-F.L.9716

FEF-F.L.9717

(eode No. FFc 3 Tg) FEF-NBM

Fi broblast-like

Mixed : Fibroblast-like and epithelial-like

Fibroblast-like

Fibroblast4 ke

Normal embryonic (3-month gestation) lung tissue of a Caucasian female Adult cotton-topped marmoset (Sagui- nus Oedipomidas) oedipus Adult white-lipped marmoset (Sugui- nus fuscicollis)

Adolescent white-lipped marmoset (S. fuscicollis)

Charles G . Rickard, New York State Veterinary College, Cornell University, Ithaca, New York Naval Biological Laboratory, Oakland, California

Newborn domestic shorthair cat

58

ASSAY OF FeSV IN CELL CULTURES

TABLE II

FOCUS ASSAY OF CO-1-ST-FeSV IN MF-69CBIM MARMOSET CELL CULTURES

Efficiency of focus formation ( %) Assay procedure First overlay Second overlay

A) Standard assay, BME-E 1 .O % bacto-agar BME-E PIUS 5 % FCS 100 plus 5 % FCS

B) BME-E PIUS 5 % FCS 1 .O % agarose BME-E PIUS 5 % FCS 52 C) BME-E PIUS 5 % FCS 1.0% ether: alcohol BME-E plus 5 % FCS 6

D) BMEI-H plus 15 % FCS 32 washed noble agar 1 .O % bacto-agar BME-E PIUS 5 % FCS

E) BME-E PIUS 5 % FCS 1 .O % bacto-agar I .O % bacto-agar 0

’ Medium used between infection and first overlay; see “Material and Methods” for complete composition of all media. Variations from the standard assay procedure are underlined.

of FeSV were added to cultures in duplicate or triplicate. The inoculated cultures were incubated for 2 h at 37” C and then fed with 5 ml of basal medium Eagle’s (BME-E) in Earle’s balanced salt solution supplemented with 5% unheated FCS and sodium bicarbonate, glutamine, peni- cillin and streptomycin in the same concentra- tions as listed for BMEI-H. After a further incubation at 37” C for 12-72 h, cultures were overlaid with 5 ml of Difco purified bacto-agar (1.25% for feline embryonic fibroblasts and 1 .O% for marmoset fibroblasts) in medium 199 supplemented with 5% unheated FCS, 0.6% tryptose phosphate broth, 0.19% sodium bi- carbonate, and 2 m ~ glutamine, 100 units penicillin and 50 pg streptomycin/ml (all given as final concentrations). The overlaid cultures were re+d 3-5 days later and weekly thereafter, without removing the basc layer of bacto-agar, with 2 ml liquid media consisting of BME-E with I% or 5% unheated FCS and the remaining additives listcd above for BME-E. Foci were countcd after various incubation periods using an inverted microscope without fixation or staining of the cultures.

Cloning of transformed cells

An agar suspension technique similar to that described by Macpherson and Montagnier (1964) was used, except that agarose was substituted for agar and 1 ml of the cell suspension (approxi- mately 7 x lo5 CAls) was seeded in 30 ml Falcon plastic T-flasks 2 to 3 days after infection with ST-FeSV.

RESULTS

Development of the focus assay

ST-FeSV induced transformation of all the human, marmoset and feline cells listed in Table I, using only fluid media, but results from experiment to experiment were irregular. In- dividual transformed cells and sometimes entire small foci became detachcd from the cell sheets soon after they first became visible and gave rise to satellite foci or scattered transformed cells throughout the cultures. Attempts therefore were made to develop a focus assay with a solid overlay and three cell cultures were selected as represen- tative marmoset and feline indicator cells: MF-69CBlM, FEF-NBM and FEF-R. The MF-69CBlM cells, which showed a uniform fibroblastic morphology, a regular growth pattern and were easy to maintain, were used in passages 10 to 18. The feline embryonic cell culture, FEF-NBM, used in passages two to four, was chosen because it was highly susceptible to FeSV in non-overlaid cultures. The feline em- bryonic fibroblast cell culture, FEF-R, selected as the second feline cell culture, was used in pas- sages 6 to 16. The different overlay procedures are listed in Table 11. The technique using a biphasic solid-fluid overlay (procedure A, Table 11) was most sensitive and gave the most consis- tent results. ST-FeSV induced foci were evident 7 to 10 days after infection of the FEF-R, FEF-NBM and MF-69CBlM cells and final focus counts were made 1% and 3 weeks after the inoculation of the FEF and M F cultures

59

MCDONALD ET AL.

respectively. No increase in the number of foci was observed when the cultures were held for an additional wcek. Foci induced by G-FeSV only became evident 15 days after infection of the cultures and focus counts were made either at that time (FEF cultures) or one week later (MF cultures).

In contrast none of the technical variations of this technique (procedures B-E, Table 11) gave comparable results. The most drastic effect on focus formation was observed when cultures were overlaid with a second layer of bacto-agar (2 ml) instead of being re-fed with fluid medium: 69CBlM cultures infected with ST-FeSV grown in either feline or marmoset cells and re-fed with bacto-agar showed no transformation, and although a few foci appeared in FEF-R cultures re-fed with bacto-agar, the foci appeared late and their number bore no relation to the virus dilution. Thus, it appears that the re-feeding of infected cultures with liquid media after the first overlay of bacto-agar is essential for the optimal development of foci.

Sensitivity and reproducibility of the focus assay

Higher titers of ST-FeSV and G-FeSV were obtained in FEF cells than in parallel assays in MF cultures. In a detailed study using CO-1- ST-FeSV, the titer was approximately four times higher in FEF cells than in MF cells (Table 111). CO-1-ST-FeSV filtrate number 2 which yielded an average titer in FEF-R of 4,129 focus forming units per ml (FFU/ml) showed an average of

only 929 FFU/ml in MF-69CBlM. The titer (FFU/ml) of a CO-I-ST-FeSV filtrate in a given cell culture was consistent from experiment to experiment (Table 111) irrespective of the pas- sage level of the cells.

Morphology of FeSV-induced foci

ST-FeSV grown in feline or marmoset cells and G-FeSV grown in feline cells generally induced foci of two morphological types in feline and marmoset cells. The foci consisted of round cells (r foci) (Fig. 1 ~ ) or of a loose meshwork of fusiform and round cells ( f r foci). On rare occasions, a third type of focus composed of only fusiforrn cells ( f foci) formed in the FEF-R cultures infected with ST-FeSV (Fig. 1 ~ ) .

Evidence from thermal stability experiments indicated that the morphological patterns r and fr were virus-dependent. Heating CO-1 -ST-FeSV at 37" C for 30 min both delayed the development of the r type foci and increased the ratio of j r / r type foci in MF cells (Table IV). Untreated virus showed an average of 158 f r and 292 r FFU/ml whereas the heat-treated virus showed an average of 254 f r and 95 r FFU/ml.

Morphology of clones of FeS V-transformed cells

The different morphological types of foci induced in MF and FEF cultures were paralleled by two morphological types of clones in CO-1- ST-FeSV infected MF-69CBl M cells grown in soft agar. One type of clone had a regular periphery and was composed of large round cells

TABLE 111

TITRATION OF CO-1-ST-FeSV IN DIFFERENT INDICATOR CELLS

Indicator cells

FFU/ml' Inoculum

Experiment (filtrate number) Experiment Experiment 1 2 3

MF-69CBIM

FEF-R

FEF-NBM

210 883 206 875

5,150 1,267

17,667 32,500

200 198 975 235 750

3,108 1,350 1,225

The FFU/ml was calculated as average number of foci/flask x dilution factor of FeSV inoculated.

60

ASSAY OF FkSV IN CELL CULTURES

FIGURE 1 Photomicrographs of foci induced by CO-I-ST-FeSV. A : r focus; B : ffocus in FEF-R cells. x 35.

TABLE IV

EFFECT OF TEMPERATURE O N r A N D fr TYPE FOCI INDUCED BY CO-1-ST-FeSV IN MF-6YCBIM CULTURES

FFU/ml Treatment of CO- I -ST-FeSV

f r Type r Type Total / r + r Ratio / r / r

Untreated . . . . . . . . 158 292 450 0.54 1 h, 25°C . . . . . . . . 256 145 402 I .75 6 h, 4°C. . . . . . . . . 248 145 383 1.71 30 niin, 37" C 254 95 348 2.67

Each FFU/mI value is the sum of the averages from two experinients, and is based on the examination of 20 flasks

(Y clones) (Fig. 2 ~ ) while the other type had a ragged periphery and was composed of irregularly shaped and small round cells ( f r clones) (Fig. 2 ~ ) .

Demonstration of a '' helper '' virus in CO-I-ST- FvS V

The titration patterns, i.e., the ratio of focus counts to virus dilutions of CO-I-ST-FeSV,

indicated that the FeSV used in these studies was either competent or that the preparations con- tained an excess of " helper " virus (Table V). The results of challenge experiments suggested that the latter is a more likely explanation.

FEF-R cultures were infected with different dilutions of CO-1-ST-FeSV. Cells treated with those dilutions of CO-I-ST-FeSV which failed to

61

MCDONALD ET AL.

FIGURE 2 Photomicrographs of clones induced by CO-1-ST-FeSV. A : r clone of 69CBlM cells composed of large

round cells; B: portion of fr clone of 69CBIM cells composed of irregularly shaped cells and small round cells. x35.

TABLE V

TITRATION OF CO-I-ST-FeSV IN MF-69CBIM AND FEF-R CELLS

FFU/ml

Dilution of MF-69CBIM FEF-R CO-1-ST-FeSV

Experiment Experiment Experiment Experiment Experiment Experiment 1 2 3 1 2 3

10" 1 :5 1:lO 1 :20 150 1 :loo 1 :500 1 :lo00

207.5 I82 275 262.5 237.5 250 725 233.5 231.5 250 750 6325 990

800 6250 1250 6550 I275 6500 1250 6375 1500

1500

' Calculated from the number of foci which developed after inoculation of given virus dilutions.

62

ASSAY OF FeSV IN CELL CULTURES

produce transformation were subcultured and were reinfected 12-14 days after the original inoculation with the same CO-1-ST-FeSV har- vest as used before. Representative data from one of three experiments are shown in Table VI. It was found that the primary inoculation with dilutions of CO-1-ST-FeSV beyond the endpoint of focus induction inhibited transformation by the second CO-1-ST-FeSV inoculation. The inhibi- tion was consistent with a 10- to 100-fold escess of a non-transforming, interfering virus (helper virus).

TABLE VI

CHALLENGE OF CO-1-ST-FeSV-INFECTED FEF-R CULTURES WITH HOMOLOGOUS VIRUS

Average number of foci/culture/0.2 ml inoculum

Dilution of original inoculum Original Challenge

inoculation (388 FFUlml) inoculation

10" 1 :10 1 :20 1 :50 1 :loo 1 :500 10-3 1 0 - 4 10-5 10-8 Control

145 16 6 4.5 0.5 0 0 0 0 0 0

N D N D N D ND 13.25 10.75 63.5 62 78.5 19 71.6

One of the non-overlaid cultures that was not transformed at a 1 :I00 dilution of CO-1-ST-FeSV was subcultured and used for challenge inoculation.

DISCUSSION

Quantitative assays measuring cell trans- formation by an oncogenic virus in cell cultures under an agar overlay were first described for Rous sarcoma viruses (RSV) by Temin and Rubin (1958). Focus assays with agar overlays (Simons et al., 19676; Zavada and Macpherson, 1970) and without agar overlays (Hartley and Rowe, 1966; Ting, 1966; O'Connor, 1968) have also been described for murine sarcoma viruses (MSV). More recently similar assay techniques without a solid overlay were used in studies of FeSV (Sarma et al., 1970; Sarma, 1971 ; Sarma and Log, 1971). In our-studies with two strains of FeSV (G-FeSV and ST-FeSV), we were unable

to obtain reproducible results in focus assays of FeSV in both feline and marmoset cell cultures with only a fluid overlay. However FeSV readily induced foci of rapidly dividing transformed cells in cultures kept under a biphasic overlay (fluid medium over a layer of solid agar) probably because this type of culture provided more optimal conditions for cell metabolism. Initial incubation under a fluid medium produced the most consistent results. It is highly unlikely that secondary foci were formed during this time because growth curve experiments showed that infectious transforming FeSV was not produced before 48-60 h after infection of FEF-R and not before 695 days after infection of MF-69CBlM cells (McDonald, unpublished data). Moreover the dose-response curve was linear in all assays in which a sufficient amount of FeSV-associated virus was present.

Feline cells were approximately four times more sensitive to focus induction by FeSV than marmo- set cells, calling to mind the lower susceptibility to RSV and MSV of cells derived from species other than the respective natural hosts (Ahlstrom et al., 1963; Bergman and Jonsson, 1962; Jensen et al., 1964; Simons et al., 1967~ ; Svoboda and Chyle, 1963 ; Zilber and Shevljaghyn, 1964).

Both ST-FeSV and G-FeSV gave rise to two morphological types of foci, similar to the foci induced by morph r and morph fr RSV in chicken cells (Temin, 1960) or MSV in mouse embryo (Hartley and Rowe, 1966) and hamster cells (Zavada and Macpherson, 1970). As in the RSV system, the morphology of the FeSV- transformed cells appeared to be determined by the virus and not by the host cell as shown by the thermal inactivation experiments. Differences in the morphology of FeSV-transformed cells were also demonstrated in cloning experiments and it is of interest that the FeSV-producing, CO-1 tumor cell line also consisted of r and f cells. It is not known whether the r or f characteristic of FeSV-transformed cells remains stable in serial cell-culture passages, and whether both r and f cells produce infectious virus. Perhaps clones of FeSV can be obtained which would induce only r , f o r fr type foci, The results of the thermal inactivation experiments may be explained by a partial inactivation of FeSV, resulting in a shift from r to f type transformation of infected cells or by a greater thermolability of clones of viruses inducing r-type transformation.

63

MCDONALD ET AL.

The presence of ‘‘ helper viruses ” in stocks of FeSV was shown recently by Sarma (1971) and Sarma and Log (1971). In our study, interference tests also indicated the presence of a 10- to 100- fold excess of non-transforming, interfering virus in the one virus preparation (CO-1 -ST-FeSV) evaluated. The fact that an associated virus is present in the CO-I-ST-FeSV preparations is interesting because this particular virus pre- paration was recovered from a tumor induced by FeSV in a non-human primate. It has not been determined, however, whether this FeSV-asso- ciated virus was present in the FeSV stock originally derived from a feline sarcoma and multiplied in the primate host together with the sarcoma virus or if it represents a primate virus acquired by the FeSV during passage in the mar- moset. Current interference and neutralization studies of all the virus preparations before and after passage in marmosets or marmoset cells should answer this question. Budding C-type particles or oncorna virus associated, group- specific antigens (gs) have not been demonstrated in our uninoculated marmoset cell cultures or in uninoculated marmosets. However in current studies it was found that both the FEF-R and FEF-NBM cell lines contain feline leukemia- sarcoma virus group-specific antigen, and budding C-type particles were demonstrated in the FEF-R

cell line (Rickard, personal communication). Most recently, we received an additional cell line from Dr. Rickard (FEF-NCE) which is free of gs antigen and C-type particles. This cell line is also susceptible to focus induction by FeSV using the biphasic overlay method (procedure A, Table 11) proving that contamination of feline cell lines with a ‘‘ helper virus ” is not a pre- requisite for FeSV-induced focus formation. Nevertheless, the apparently frequent presence of C-type viruses containing gs antigen (feline associated viruses, FeAV) in cell lines derived from ‘‘ normal ” embryonal or newborn feline tissues must be considered in studies with feline leukemia virus (FeLV) and FeSV. The presence of FeAV may influence results of studies on the genetic susceptibility of feline cells to various strains of FeLV or FeSV, or distort antigenic characteristics of various virus isolates and the interference patterns among them.

ACKNOWLEDGEMENTS

This study was supported by research contract No. NIH-71-2032 within the Special Virus Cancer Program of the National Cancer Institute, and by General Research Support Grant No. RR-05477, from the National Institutes of Health, US Public Health Service.

VIRUS DU FIBROSARCOME FELIN: TEST QUANTITATIF SUR LES FOYERS, MORPHOLOGIE DES FOYERS ET PREUVE DE LA PRESENCE

D’UN ‘‘ VIRUS D’APPOINT ”

Le virus du Jibrosarcome fklin ( VSFe) a ktP testi par la formation de foyers dans des cultures de jibroblastes de chat et de ouistiti couvertes d’une couche de gilose et recouvertes de milieu liquide. Les titres de VSFe concordaient d’une expkrience a l’autre et, lors de tests parall2les, ils ktaient environ quatre fois plus klevks pour les Jibroblastes de chat que pour ceux de ouistiti. Les clones de cellules infectkes par le VSFe cultivks dans la gilose molle et les foyers de cellules transformies produits par le VSFe dans des cultures cellulaires en couche unique relevent de deux types rnorphologiques: des cellules rondes ( r ) et un rkseau lache de cellules firsiformes et rondes ( f r ) . Des experiences de stabilitk thermique ont montre que les differetices morphologiques observees au niveau des foyers dkpendent du virus.

Le plan de titrage du VSFe rPcoltk a partir de celhles de chat ou dc ouistiti montre que ces prkparations sont opposers ou qu’elles contiennent un exces de virus d’appoint. Des expiriences prdiminaires de confrontation avec la souche Snyder- Theilen du VSFe ricolrke dans des cellules de ouistiti indiquent qu’un virus interfirent (virus d’appoint) non transformateur est present dans cette preparation de VSFe dans une proportion de I 0 a 100 fois suptrieure a celle de VSFe.

64

ASSAY OF FeSV IN CELL CULTURES

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