described by different authors (Berman & Allison, 1969; Dalton, 1966; Perk ... (grade + 1) after 4-10 days, and then either died (10-13 days post inoculation).
Laboratory Animals (1973) 7, 255-263.
255
TUMOUR REGRESSION AND RELAPSE IN MICE INOCULATED WITH MURINE SARCOMA VIRUS (MOLONEY) by K. PERK*, E. RUSSELL, K. L. SMITH
AND
J. B. MOLONEY
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. SUMMARY
The oncogenic effect of the virus was tested in mice of different strains and ages. Local sarcomas were induced in all mice, although strain, age or dose dependent variations were found. 4 forms of development occurred: progressive lethal; lethal but long persistent; complete tumour regression; tumour recurrence after regression. Virus activity was highest in the progressively-growing and in the recurrent tumours, while in the longpersistent tumours or in tissue at the site of regressed tumours, little or no virus could be detected. TUMOR REGRESSION UNO RUCKFALL IN MAUSEN, DIE MIT MURINE SARCOMA VIRUS (MOLONEY) GEIMPFT SIND
Die onkogenische Wirkung des Virus wurde in Mausen verschiedener SHimme und verschiedenen Alters gepriift. Lokale Sarkome wurden in allen Mausen hervorgerufen, obgleich Unterschiede, von Stamm, Alter und Dosis abhangig, gefunden wurden. Es gab 4 Arten der Entwicklung: Progressiv lethal; lethal, aber lange-persistent; v611ige Tumorregression; Wiederauftreten des Tumors nach Regression. Virus Aktivitat war am Starksten in den progressiv wachsenden und in den wiederauftretenden Tumoren, wahrend in den lange-persistenten Tumoren oder in Geweben an Stelle von ri.'lckgebildeten Tumoren wenig oder kein Virus entdeckt werden konnte. REGRESSION DES TUMEURS ET RECHUTE DANS LES SOURIS VACCINEES AVEC MURINE SARCOMA VIRUS (MOLONEY)
L'effet oncologique du virus fut examine dans des souris de souches et d'ages differents. Des sarcomes locals furent induits dans toutes les souris, bien qu'il y avait des variations selon les souches, l'age et la dose. 4 formes de developpement furent observees: progressive lethale; lethale mais longuement persistante; regression totale des tumeurs; tumeur *VisitingScientist: Hebrew University of Jerusalem, Rehovot Campus, Israel.
256
K. PERK,
E. RUSSELL,
K. L. SMITH
AND
J. B. MOLONEY
recurrent apres regression. L'activite du virus etait au plus fort dans les tumeurs de developpement progressif et recurrent, tandis que dans les tumeurs longuement persistants et dans Ie tissu a I'endroit de tumeurs involus on trouvait peu Oll point de virus. The histopathology and ultrastructure of tumours induced in mice by various preparations of the murine sarcoma virus (Moloney)-MSV(M)-have been described by different authors (Berman & Allison, 1969; Dalton, 1966; Perk & Moloney, 1966; Perk & Hod, 1971; Siegler, 1970; Simons & McCully, 1970; Stanton, Law & Ting, 1968). In this study the MSV(M)-induced tumour was characterized and classified as an undifferentiated sarcoma (Perk & Hod, ]971). A unique feature of this neoplasm is a high incidence of complete regression of the local tumour in immunologically competent mice (Blumenschein & Moloney, 1969; Fefer, McCoy & Glynn, 1967; Fefer, McCoy, Peark & Glynn, 1968; Gazdar, 1970; Law, Ting & Stanton, 1968; Schlom, Moloney & Groupe, 1970). Little documentation is available on the fate of the mice after tumour regression (Gazadar, 1970; Schlom et at., 1970). This report examines and correlates the rate of tumour regression and relapse to the age and strain of mice, and to the dose of virus inoculum used in a long-term experiment. MA TERIALS
AND METHODS
A total of 648 mice from 7 different strains-DBA/2N, AKR/N, AL/N, C3H/HeN, N:GP(SW), and BALB/c-were used in this study. They were composed of 3 age groups: newborn, 4- and 6-week-old. The newborn were inoculated with 0·1 ml of virus preparations while the 4- and 6-week-old were, in addition, inoculated with 0-2, 0-4, or 0,8 ml of the same preparation. If not otherwise stated, the groups were composed of 8 or more animals of both sexes per treatment. They were housed in stainless-steel cages and fed pelleted laboratory diet and water ad libitum. The tumour response was determined and graded on an arbitary scale of + 1 to +4 (Blumenschein & Moloney, 1969). The experiment was ended after 400 days. The animals were dissected after they died or were killed, and samples were taken for histology. The MSV(M) mouse-derived preparation used was previously passaged in BALB/c mice for 152, 153, and 154 generations. The tumours were weighed and a cell-free extract was prepared by differential centrifugation (Moloney, 1960). The preparations were pooled and adjusted so that 1 g of tumour tissue provided 1 ml of extract. Virus activity per 1 g tumour showed a ED 5 0 of 10-4•5 as tested in newborn BALB/c mice. The virus preparation (either 0'1, 0'2, 0,4, or 0,8 ml) was injected by the subcutaneous-intramuscular route (Perk, Moloney & Jekins, 1967).
C57BL/N,
MSY TUMOURS
257
IN MICE
RESULTS
Tumour induction The MSY(M) preparations used induced local sarcomas in all 648 mice (100 % incidence), regardless of strain, sex, age, or dose tested (Table I). There was some variation in time to the development of a +4 grade tumour-3-4 days in the newborn and 4-week-old animals, 5-9 days in the 6-week-old animals. Exceptions were the 6-week-old AKRjN mice inoculated with 0·2 ml or 0·4 ml of virus material, which developed only small nonprogressive tumours (grade + 1) after 4-10 days, and then either died (10-13 days post inoculation) or their tumours regressed (Figs I & 2). The AKRjN mice inoculated with 0·8 ml developed large (+4) progressively-growing lethal tumours after a long latent period (70 days). Table 1. Mortality from primary tumours and from relapse after tumour regression in newborn and 4-week-old mice of different strains. The mice were inoculated with 0·1 ml of 1 g equivalent virus preparations. Newborn Strain
mice inoculated*
died with lary tumour
4-week-old mean time to death (days)
mice inoculated*
dead with lary tumour
mice with regressed tumours
dead with regressed tumour
mean time to death
(days) DBA
15
15
8
10
0
10
10
94
AKRjN
12
12
6
10
I
9
8
180
8
8
6
10
1
9
4
320 120
ALfN
8
8
7
10
0
10
4
C3HjHeN
15
15
8
10
I
9
0
N:GPj(SW)
15
15
8
0
BALBjc
14
14
7
10
1
9
5
C57fBL
159
*AII mice inoculated developed tumours.
Tumour regression and recurrence While there was no strain or age difference in the incidence of tumour induction, definite host variations in 'tumour fate', i.e. tumour progression, regression or relapse, were observed. Age. All newborn mice of all the strains tested, developed progressivelygrowing lethal tumours (Table I). By contrast, in the 4- and 6-week-old mice a high incidence of tumour regression (15-40 days post inoculation) was seen (Figs I & 2, Table I). However, in a large percentage (Figs 1 & 2, open bars, and Table I) the tumours appeared at the site of inoculation where
258 K. PERK, E. RUSSELL, K. L. SMITH
AND
--,,-,
J. B. MOLONEY
BARS DOSE (mil
100 (7)""
-
NID
3rd
\\lID 1Il-
., N
.0.,
80
~
0.2 0.4 0.8
2nd
--
III
f'o.lD
60
,... ID
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lD (7)
.0 N
.,.,
(7) CD
...J
«
I-
a:: ~ 40
20
o
....
:.:.:. :.:,',
AKRIN
ALIN
STRAIN Fig. 1. Mortality from primary tumour (shaded bars) and from relapse after tumour regression (open bars) in various strains of mice, inoculated at the age of 4 weeks with different dosages of MSV(M). The numbers in the bars indicate mean time, in days, to death with tumours.
the initial inoculum did not produce a rapidly-growing tumour, but some tumours appeared at a later date at sites unrelated to those of the primaries. In most of these cases where tumours appeared at a later date animals showed tumour nodules in the lungs, but tumours on or in the diaphragm, body-wall musculature, kidney, spleen and liver were also common. Many animals with tumours had also large amounts of ascites fluid. The infiltrating nodules were similar to the initial tumour at the subcutaneous or intramuscular site. The times to death (in days) of the host animals from the primary tumour
MSV TUMOURS IN MICE (shaded bars) or of relapse after the initial tumour indicated in Figs I & 2. BAAS -I ., 100
-
-
., CD
•....
0 ,....
lD
regression (open bars) are
DOSE ( m I)
0.2 0.4 0.8
2 nd
-
259
3rd
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., -
., -N
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