Symposium on Immunotherapy in Malignant Disease
Modified Tumor Cells in the Immunotherapy of Solid Mammary Tumors Richard L. Simmons, M.D.,* Angelyn Rios,** and Paul Trites, B.8.***
We previously demonstrated that methylcholanthrene (MCA)induced fibrosarcomas transplanted into syngeneic C3H/HeJ female recipients could be made to regress if the mice are challenged with tumor cells treated in vitro with Vibrio cholerae neuraminidase (VCN).36 The regression is immunospecific. 36 The immunoregressive effect could be augmented by simultaneous inoculations of nonspecific immunostimulants, e.g., Mycobacterium bovis strain BCG,32, 34 and the effect was not abrogated by pretreatment of the challenging tumor cells with mitomycin C to prevent the growth of the challenging inoculum. The present studies were designed to determine whether the immunoregression of transplantable and spontaneous mammary tumors could be induced by challenge with VCN-treated turn or cells.
MATERIALS AND METHODS Mice C3H/HeJ and C3HeB/FeJ female mice were obtained from Jackson Laboratory, Bar Harbor, Maine. The animals were housed separately in covered plastic cages, 10 or less per cage, and provided with tap water and a standard laboratory diet (Purina Chow Checkers) ad libitum. C3H/HeJ mice are infested with a mammary tumor virus (MTV) and will develop a high incidence of spontaneous mammary tumors in the second year of
"Professor of Surgery and Microbiology, University of Minnesota, Minneapolis **Principal Laboratory Technician, Department of Surgery, University of Minnesota ***Junior Scientist, Department of Surgery. University of Minnesota This work was supported by U.S. Public Health Service Grant No, 11605 and Grant IM-20A from the American Cancer Society.
Medical Clinics of North America-Vol. 60, No. 3, May 1976
551
552
RICHARD
L.
SIMMONS, ANGELYN RIOS, AND PAUL TRITES
life. C3HeB/FeJ is a strain of mice derived from C3H/HeJ ova transferred to C57 BL/6 mice by Fekete in 1949. C3HeB/FeJ mice are believed not to contain the same mammary tumor virus which infests the C3H/HeJ strain since they have a lower incidence of mammary tumors. 7 , 11
Tumors Several spontaneous mammary adenocarcinomas (M-I, M-2) appearing in aged C3H/HeJ mice were maintained by serial transplantation in syngeneic C3H/HeJ female mice. Sterile tumor cells for inoculation were prepared from tumors (1.0 to 1.4 cm in diameter) growing in C3H/HeJ female mice 3 to 4 weeks following inoculation. Cells were pressed through No. 45 mesh stainless steel screens in Medium 199 (MI99) (Grand Island, New York) without addition of trypsin. Cell clumps were then allowed to settle and the supernatant single cell suspensions were washed three times in M199 and counted in hemocytometers. Viability was determined by trypan blue exclusion. 2 In transplanted tumor experiments, the living tumor cells were injected into the subcutaneous tissue of the lateral posterior flank ofrecipient C3H/HeJ or C3HeB/FeJ mice. The largest diameter of the growing tumor was measured by calipers three times weekly and the day of death was also recorded. Neuraminidase Vibrio cholerae neuraminidase (VCN) obtained from Behring Diagnostics (Somerville, New Jersey) is stated to contain 500 u enzyme per ml. (One unit of activity is equivalent to the release of 1 /J-g of N-acetyl neuraminic acid from a glycoprotein substrate at 37° in 15 minutes at pH 5.5). VCN is inactivated by heating to 65°C for 30 minutes or 100°C for 10 minutes. Sialic acid is released from cell surfaces of normal and malignant cells by VCN at neutral pH without affecting viability of the cells.1, 3, 9, 20,25, 30,42 Tumor cells in M199 (pH 7.2) were incubated for 1 hour at 37°C with 25 u VCN per ml per 10 6 cells, with heat-inactivated VCN, or with M199. The cells were washed three times prior to injection as a challenging inoculum into tumor-bearing hosts. Mitomycin C Mitomycin C was obtained from Sigma Chemical Company (St. Louis, Missouri) and incubated with tumor cells in a concentration of 25 /J-g per ml per 106 cells in the same incubation mixture with VCN or heat-inactivated VCN. We have previously demonstrated that mitomycin C has no adverse effect on the efficacy of VCN-treated cells to induce immunospecific regression of firmly established methylcholanthrene fibrosarcomas in C3H/HeJ mice. Treatment of tumor cells with mitomycin C does not increase the uptake of trypan blue by such tumor cell suspensions, but effectively prevents the growth of palpable tumors from the treated cells. Transplantable Tumor Experimental Protocol The basic experimental design in transplantable tumor experiments was to inoculate adult female C3H/HeJ mice or C3HeB/FeJ mice with
MODIFIED TUMOR CELLS IN SOLID MAMMARY TUMORS
553
viable tumor cells subcutaneously into the left lateral posterior flanks. The tumors usually reached palpable size by day six and measured 0.3 to 0.9 cm by day 15. The tumors were then either left untreated or excised, and the host was challenged with VCN-treated cells into the subcutaneous tissues of the right flank. The challenging inoculum was then repeated on alternate days to a total of six inoculations. The effect ofVCNtreated tumor cells on the growth of the firmly established mammary tumors was then determined. Spontaneous Tumor Experimental Protocol Our experiments utilizing spontaneous tumors were somewhat different. Adult female C3H-HeJ mice naturally infected with the mammary tumor virus were utilized. Mice were purchased when they were 9 to 15 months old and after having delivered multiple litters at the Jackson Laboratories. The mice were examined twice weekly for the appearance of spontaneous mammary adenocarcinomas. When the tumors were found (0.7 to 0.9 cm in diameter) the mice were individually placed in precoded cages. A second investigator then utilized the code to determine the tumor treatment to be administered. Two experiments were designed. In the first, the tumor treatment consisted of intratumor injections every fourth day with 0.1 ml sterile solution containing M199, 50 u of VCN, heat-inactivated VCN, 1 mg Mycobacterium bovis (strain BCG), or a combination of VCN, heatinactivated VCN, and/or BCG. Tumor diameter was measured every 4 days just prior to next treatment, and the date of death recorded. In the second set of experiments, designed to treat spontaneous mammary tumors in retired breeding C3H/HeJ female mice, surgical excision was combined with VCN or inactivated VCN-treated autochthonous or M-I cells. When the spontaneous tumors were found, the mice were individually placed in precoded cages. Fourteen days later, when progressive tumor growth was assured, tumor excision and immunotherapy was carried out according to the cage code. The immunotherapeutic code was not revealed until after excision was performed in order to eliminate bias in the performance of the surgery. Effect of VCN-Treated Mammary Carcinoma Cells on the Growth of Established Transplantable Mammary Adenocarcinomas in Syngeneic C3H/HeJ Hosts In the left flank of recipient syngeneic C3H/HeJ mice, 40,000 M-I transplantable mammary adenocarcinoma cells were injected subcutaneously. Fifteen days thereafter, when the tumor nodules measured 0.3 to 0.8 cm in diameter, recipient mice were inoculated with 106 M-I tumor cells that had been incubated either with VCN (plus mitomycin) or heat-inactivated VCN (plus mitomycin). Results are summarized in Table 1. The injection of 10 6 M-I tumor cells exposed to heat-inactivated VCN did not affect the progressive fatal growth ofthe tumors. In contrast, injection of 106 VC N -treated M-I cells markedly slowed tumor growth and produced total regression of 4/40 firmly established M-I mammary adenocarcinomas. Animals whose tumors totally disappeared survived indefinitely, and life was significantly prolonged even in those animals
554
RICHARD
L. SIMMONS, ANGELYN Rws, AND PAUL TRITES
Table 1. Effect ofVCN-Treated M-l Mammary Adenocarcinoma Cells on Regression of M-l Tumors in C3HIHe] Mice
GROUP
TREATMENT OF CELLULAR CHALLENGE*
FRACTION OF PRIMARY TUMORS UNDERGOING TOTAL REGRESSION
MEAN TUMOR DIAMETER DAY 30 (cm ± SE)
MEAN DAY OF DEATH (± SE)**
2 3
VCN Inactive VCN
0/30 4/40 0/40
1.89 ± 0.09 1.06 ± 0.10*"* 1. 79 ± 0.07
49.4 ± 2.2 60.4 ± 3.3*** 49.3 ± 2.1
'Challenging inocula consisted of 6 injections of 106 M-I cells incubated with VCN (25 U/ml/10 6 cells) or heat-inactivated VCN (plus mitomycin C). First challenge was given 15 days after tumor inoculation and subsequent challenges at 2-d ay intervals. "*Does not include mice whose tumors totally regressed. ***p ., 0.05 when compared to groups 1 and 3 by Student's t test.
whose tumors did not totally disappear. A similar experiment was carried out with a second mammary adenocarcinoma (M-2) (Table 2). Two experiments were then done to determine the immunospecificity of the regression induced by VCN-treated mammary adenocarcinoma cells. The first such experiment is summarized in Table 3. Into the left flank of syngeneic C3H/HeJ mice, 40,000 M-I mammary adenocarcinoma cells were injected subcutaneously. Fifteen days later, tumorbearing mice were challenged with 10 6 M-I or 10 6 M-2 mammary adenocarcinoma cells that had been exposed to VCN (plus mitomycin) or heat-inactivated VCN (plus mitomycin). Slowing of tumor growth and prolongation of life in mice with M-I tumors were seen only when injections of M-I tumor exposed to VCN were given. Total regression in this series occurred in 1 of 20 animals. Tumor growth was not inhibited in recipients challenged with M-2 tumors or recipients challenged with M-I tumors treated with inactivated VCN (Table 3).
Cross-Immunization of M-I Tumors with M-2 Tumors in C3HeB/FeJ Mice These results suggested that M-I and M-2 did not share the same tumor-specific transplantation antigens. We needed to confirm this fact Table 2. Effect ofVCN-Treated M-2 Mammary Adenocarcinoma Cells on Regression of M-2 Tumors in C3HIHe] Mice
GROUP
TREATMENT OF CELLULAR CHALLENGE*
FRACTION OF PRIMARY TUMORS UNDERGOING TOTAL REGRESSION
MEAN TUMOR DIAMETER DAY 30 (cm ± SE)
MEAN DAY OF DEATH (± SE)"
1 2 3
VCN Inactive VCN
0/10 1110 0/10
1.24 ± 0.04 0.51 ± 0.08*'" 1.07 ± 0.04
78.0 ± 2.3 94.3 ± 3.7*** 72.4 ± 2.2
'Challenging inocula consisted of 6 injections of 106 M-2 cells incubated with VCN (25 U per mI per 106 cells) or heat-inactivated VCN (plus mitomycin C). First challenge was given 15 days after tumor inoculation and subsequent challenges at 2-day intervals. **Does not include mice whose tumors totally regressed. ***p ., 0.05 when compared to groups 1 and 3 by Student's t test.
555
MODIFIED TUMOR CELLS IN SOLID MAMMARY TUMORS
Table 3. Effect ofVCN-Treated M-l or M-2 Cells on Regression of M-l Tumors in C3HIHe] Mice
GROUP
CHALLENGING TUMOR
TREATMENT OF CELLULAR CHALLENGE*
FRACTION OF PRIMARY TUMORS UNDERGOING TOTAL REGRESSION
1 2 3 4 5
M-I M-I M-2 M-2
VCN Inactive VCN VCN Inactive VCN
0/20 1120 0/20 0/20 0/20
MEAN TUMOR DIAMETER, DAY 30 (cm ± SE) 1. 73 ± 1.03 ± 1.79 ± 1.81 ± 1.82 ±
0.10 0.12*** 0.10 0.08 0.09
MEAN DAY OF DEATH (± SE)** 51.0 66.8 52.7 53.5 51.4
± ± ± ± ±
3.8 4.8*** 3.3 3.2 3.2
*Challenging inocula consisted of 6 injections of M-I or M-2 cells incubated with VCN (25 U per ml per 106 cells) or heat-inactivated VCN (plus mitomycin C). First challenge was given 15 days after tumor inoculation and subsequent challenges at 2-d ay intervals. ':'*Does not include animals whose tumors totally regressed. **"p ~ 0.05 when compared to groups 1, 3, 4, and 5 by Student's t test.
before proceeding further. Therefore, 106 M-I or M-2 viable tumor cells from C3H/HeJ mice were injected subcutaneously in the left posterior flank of adult female C3HeB/FeJ mice. All injected mice developed tumors. Control mice received 106 C3H/HeJ or C3HeB/FeJ spleen cells or an equal volume (0.1 ml) of M199. Two weeks following the injection, when the tumors ranged in diameter from 0.8 to 1.6 cm, the tumor masses were totally excised, and excision of normal tissues were performed in mice not bearing tumors. Two weeks after tumor excision (one month after tumor inoculation), all mice were challenged in the subcutaneous tissues of the right posterior flank with 5x 10 3 , 5x 104 , or 5x 105 M-I orM-2 viable tumor cells. The results are recorded in Table 4. C3HeB/FeJ mice who had not received previous injections of M-I or M-2 turn or cells developed tumors from the challenging inocula which progressed to death (Table 4). C3HeB/FeJ mice pre-exposed to nonmalignant C3H/HeJ or C3HeB/FeJ spleen cells supported a similar proportion of either M-I or M-2 tumors. C3HeB/FeJ mice immunized with either M-I or M-2 tumors failed to support both M-I or M-2 tumors (P ,,; 0.05) although it seemed that mice pre-exposed to M-2 tumors supported a significantly smaller proportion of either M-I or M-2 tumors than did mice pre-exposed to M-I tumors (p ,,; 0.05). These results strongly suggest that M-I and M-2 tumors share the same mammary tumor virus tumor antigen, and that C3HeB/FeJ mice can be immunized by the mammary tumor virus antigen from the C3HI HeJ mammary tumors (Table 4). Effect of VCN-Treated Mammary Carcinoma Cells on the Growth of Simultaneous Established M-I and M·2 Tumors in Syngeneic C3H/HeJ or C3HeB/FeJ Mice 40,000 viable M-I tumor cells were injected subcutaneously into the left flank of C3H/HeJ or C3HeB/FeJ mice. Simultaneously 40,000 M-2 cells were injected in the right flank of the same mice. Thus, separate M-I and M-2 tumors were established in both the mammary tumor virus-
556
RICHARD
L.
SIMMONS, ANGELYN RIOS, AND PAUL TRITES
Table 4. Growth of M-l or M-2 Mammary Carcinomas in C3HeBIFe] Mice Pre-exposed to M-l or M-2 Tumors PRIMARY INOCULUM
None
C3H/HeJ
Spleen Cells
C3HeB/FeJ
Spleen Cells
CHALLENGING
NO. OF
FRACTION OF MICE SUPPORTING
TUMOR
CELLS
SECONDARY TUMOR
M-I
5 x 103 5 x 104 5 x 10"
3/20 14/20 20/20
M-2
5 x 103 5 x 104 5 x 10"
3/20 15/20 20/20
M-I
5 x 103 5 x 10' 5 x 10"
1/10 6/10 10/10
M-2
5 x 103 5 x 104 5 x 10"
7/10 10/10
5 x 103 5 x 104 5 x 10"
7/10 10/10
5 x 103 5 x 10' 5 x 10"
8/10 10/10
M-I
M-2
M-I
M-I
M-2
M-2
1/10
1/10
1/10
5 x 103 5 x 104 5 x 10"
2110
5 x 103 5 x 104 5 x 10"
2/10
1/10
4/10
3/10 4/10
M-I
5 x 103 5 x 104 5 x 10"
0/20
M-2
5 x 103 5 x 104 5 x 10"
0/20 0/20
1/20 1/20
1/20
infested C3H/HeJ mice and in the mammary tumor virus-free C3HeB/FeJ mice. Twelve days thereafter, after separate tumors were palpable, the mice were challenged at a third site with either 10 6 M-I or M-2 tumor cells which had been exposed to VCN (plus mitomycin) or inactivated VCN (plus mitomycin). The regression of the pre-existing tumors was then determined. The results are shown in Table 5. Inhibition of growth and regression of firmly established M-I tumors took place only in animals challenged with VCN-treated M-I tumors. M-I tumors did not regress when mice were challenged with M-I tumors exposed to inactivated VCN, nor did the M-I tumor regress when treated with M-2 cells exposed
557
MODIFIED TUMOR CELLS IN SOLID MAMMARY TUMORS
Table 5. Immunospecificity of Mammary Tumor Regression in Viral-Infested (C3H/HeJ) or Viral-Free (C3HeBIFe]) Mice Bearing Both M-l and M-2 Tumors TUMOR BEARING HOST e3H/HeJ
e3HeB/FeJ
GROUP NO.
TREATMENT OF CHALLENGING INOCULUM"
FRACTION TUMORS REGRESSING M-1 b M_2b
1 2 3 4 5
None M-I M-I M-2 M-2
M199 VeN Inactive VeN VeN Inactive VeN
0/14 3/20 0/17 0/19 0/15
0/14 0/20
6 7 8 9 10
None M-I M-I M-2 M-2
M199 VeN Inactive VeN VeN Inactive VeN
0110 6/19 0/18 0/20 0/19
MEAN TUMOR DIAMETER DAY 30 (cm ± SE) M-I M-2
0/15
0.90 0.40 0.86 1.07 1.11
± ± ± ± ±
0.07 0.05' 0.04 0.06 0.05
1.20 ± 1.32 ± 1.19 ± 0.52 ± 1.46±
0.06 0.06 0.06 0.08** 0.07
0/19 0/19 0/18 4/20 0/19
1.09 0.37 0.87 0.96 0.94
± ± ± ± ±
0.05 0.06**0.03 0.04 0.04
1.06± 1.11 ± 1.03 ± 0.42 ± 1.22 ±
0.03 0.04 0.04 0.07***0.04
0117 1119
*p ,,; 0.05 when compared to groups 1, 3, 4, 5, and contralateral tumor. **p,,; 0.05 when compared to groups 1,2, 3, 5, and contralateral tumor. ***p ,,; 0.05 when compared to groups 6, 8, 9, 10, and contralateral tumor. ****p ,,; 0.05 when compared to groups 6, 7, 8, 10, and contralateral tumor. aehallenging inocula consisted of six injections of M-I or M-2 cells which had been incubated with 25 u/ml/106 cells of veN or heat-inactivated veN (plus mitomycin e). The first challenge was given 12 days after tumor inoculation and subsequent challenges at two day intervals. bDoes not include animals whose tumors blended so that each could not be separately distinguished.
to VCN or inactivated VCN. Conversely, M-2 tumors did not regress if mice were challenged with M-2 tumors exposed to inactivated VCN, or to M-I cells in any form. The strain of the host mouse had no effect on this response. Even the C3HeB/FeJ mice free of the mammary tumor virus failed to reject M-I tumors when challenged with VCN treated M-2 cells, and vice versa. Effect of N euraminidase-Treated Mammary Adenocarcinoma Cells on the Regression of Partially and Totally Excised Adenocarcinomas in C3H/HeJ Mice 80,000 M-2 mammary adenocarcinoma cells were injected subcutaneously into the left flank of recipient C3H/HeJ mice. Fifteen or 25 days thereafter, wedge excision, enucleation, or total tumor excision was carried out and the mice were challenged with 10 6 living M-2 tumor cells which had been incubated either with VCN or heat-inactivated VCN (plus mitomycin). The results are tabulated in Table 6. Mice challenged on either day 15 or day 25 with VCN-treated tumor cells demonstrated significant slowing oftumor growth. Wedge excision of the tumor on day 15 added nothing to the immunoregressive effect of VCN-treated tumor cells. One of the 10 tumors disappeared even without tumor excision. When treatment was delayed until day 25, tumor re growth was significantly inhibited by a combination of partial, subtotal, or attempted
Total Total
25 25
VCN Inactive VCN
7/10 3/10
1110 0110
0/10 0/10 0/10
0/10 0/10
0/10 1110 0/10
REGRESSING
0.61 ± 0.05 0.63 ± 0.06 0.00 ± 0.00 0.00 ± 0.00
0.91 ± 0.03 0.92 ± 0.03
1.19 ± 0.06 1.08 ± 0.06 1.11 ± 0.04
0.96 ± 0.05 0.95 ± 0.04 0.91 ± 0.04 0.95 ± 0.05 0.90 ± 0.03
0.27 ± 0.02 0.30 ± 0.02
0.29 ± 0.03 0.30 ± 0.03 0.31 ± 0.03
24 hr post excision
0.25 ± 0.02 0.28 ± 0.03
0.27 ± 0.03 0.26 ± 0.03 0.28 ± 0.03
24 hr prior to excision
SE)
2 weeks post excision
±
0.14 ± 0.07"'* 0.33 ± 0.16**
0.98 ± 0.13**t 2.22 ± 0.18
2.61 ± 0.19 1.85 ± 0.17 2.78 ± 0.09
0.86 ± 0.15* 1.03 ± 0.21
1.47 ± 0.09 0.56 ± 0.10* 1.44 ± 0.08
MEAN TUMORDIAMETER (cm
SE)b
103.7 ± 13.0**t 77.6 ± 13.5*'"
87.0 ± 4.4**t 65.0 ± 3.5
60.9 ± 4.9 74.7 ± 3.1**t 59.8 ± 3.0
61.0±5.7 59.7 ± 5.1
60.9 ± 4.9 65.9 ± 5.9 64.0 ± 4.2
(±
OF DEATH
MEAN DAY
6
aChallenging inocula consisted of six injections of 10 M-2 cells, the first challenge was given 15 or 25 days after tumor inoculation with subsequent challenges at 2 day intervals. bDoes not include mice whose tumors totally regressed. *p ,;; 0.05 when compared to groups 1, 3, and 5 (Student's T test). "'*p ,;; 0.05 when compared to groups 6, 8, and 10 (Student's T test). tp ,;; 0.05 when compared with mice challenged with inactive VCN treated cells (Student's T test). ttGroups 1 and 6 refer to the same animals, tumors being measured at differing intervals after implantation.
12
11
VCN Inactive VCN
Enucleation Enucleation
25 25
9 10
VCN Inactive VCN
None None None
25 25
6tt 7 8
VCN Inactive VCN
Wedge Wedge
15 15
4 5
VCN Inactive VCN
15 15
Ht 2 3
None None None
(DAYS)
NO.
CHALLENGE a
CELLULAR
TREATMENT
GROUP
EXCISION
PRIMARY TUMORS
TREATMENT OF
IMPLANTATION AND TOTALLY
FRACTION OF
INTERVAL BETWEEN
Table 6. Effect of Tumor Excision and Immunotherapy on the Growth of M-2 Tumors in C3HIHej Mice
'"
t'l
::l
;;;l
t"'
~
"ti
Ij
~
'"
"" o
::0
:z
~ ~
'" > :z
~
~
r:tJ
t:""
~
>
~
::0
"" n
U< U< CXJ
559
MODIFIED TUMOR CELLS IN SOLID MA~MARY TUMORS
total excision plus challenge with VCN-treated tumor cells. Seven of 10 animals were cured by at~empted total tumor excision plus VCN-treated tumor cells. Partial tumor excision plus challenge with heat-inactivated VCN -treated tumor cells did not significantly inhibit tumor regrowth, and total excision plus inaetivated VCN-treated, tumorcells cured on\y three of 10 animals. Effect of Direct In.~ra~umor Injections of Neuraminidase ancJ/or BCG into Spontaneous Mammary Adenocarcinomas in C3H/HeJ Mice It is possible th:1lit transplantable mammary tumors do not resemble spontaneous mammary tumors in their responses to immunotherapy with neuraminidase-treated cells. Since we had shown that the repeated intralesional injection of-VGN into transplantable fibrosarcomas le
Modified Tumor Cells in the Immunotherapy of Solid Mammary Tumors Richard L. Simmons, M.D.,* Angelyn Rios,** and Paul Trites, B.8.***
We previously demonstrated that methylcholanthrene (MCA)induced fibrosarcomas transplanted into syngeneic C3H/HeJ female recipients could be made to regress if the mice are challenged with tumor cells treated in vitro with Vibrio cholerae neuraminidase (VCN).36 The regression is immunospecific. 36 The immunoregressive effect could be augmented by simultaneous inoculations of nonspecific immunostimulants, e.g., Mycobacterium bovis strain BCG,32, 34 and the effect was not abrogated by pretreatment of the challenging tumor cells with mitomycin C to prevent the growth of the challenging inoculum. The present studies were designed to determine whether the immunoregression of transplantable and spontaneous mammary tumors could be induced by challenge with VCN-treated turn or cells.
MATERIALS AND METHODS Mice C3H/HeJ and C3HeB/FeJ female mice were obtained from Jackson Laboratory, Bar Harbor, Maine. The animals were housed separately in covered plastic cages, 10 or less per cage, and provided with tap water and a standard laboratory diet (Purina Chow Checkers) ad libitum. C3H/HeJ mice are infested with a mammary tumor virus (MTV) and will develop a high incidence of spontaneous mammary tumors in the second year of
"Professor of Surgery and Microbiology, University of Minnesota, Minneapolis **Principal Laboratory Technician, Department of Surgery, University of Minnesota ***Junior Scientist, Department of Surgery. University of Minnesota This work was supported by U.S. Public Health Service Grant No, 11605 and Grant IM-20A from the American Cancer Society.
Medical Clinics of North America-Vol. 60, No. 3, May 1976
551
552
RICHARD
L.
SIMMONS, ANGELYN RIOS, AND PAUL TRITES
life. C3HeB/FeJ is a strain of mice derived from C3H/HeJ ova transferred to C57 BL/6 mice by Fekete in 1949. C3HeB/FeJ mice are believed not to contain the same mammary tumor virus which infests the C3H/HeJ strain since they have a lower incidence of mammary tumors. 7 , 11
Tumors Several spontaneous mammary adenocarcinomas (M-I, M-2) appearing in aged C3H/HeJ mice were maintained by serial transplantation in syngeneic C3H/HeJ female mice. Sterile tumor cells for inoculation were prepared from tumors (1.0 to 1.4 cm in diameter) growing in C3H/HeJ female mice 3 to 4 weeks following inoculation. Cells were pressed through No. 45 mesh stainless steel screens in Medium 199 (MI99) (Grand Island, New York) without addition of trypsin. Cell clumps were then allowed to settle and the supernatant single cell suspensions were washed three times in M199 and counted in hemocytometers. Viability was determined by trypan blue exclusion. 2 In transplanted tumor experiments, the living tumor cells were injected into the subcutaneous tissue of the lateral posterior flank ofrecipient C3H/HeJ or C3HeB/FeJ mice. The largest diameter of the growing tumor was measured by calipers three times weekly and the day of death was also recorded. Neuraminidase Vibrio cholerae neuraminidase (VCN) obtained from Behring Diagnostics (Somerville, New Jersey) is stated to contain 500 u enzyme per ml. (One unit of activity is equivalent to the release of 1 /J-g of N-acetyl neuraminic acid from a glycoprotein substrate at 37° in 15 minutes at pH 5.5). VCN is inactivated by heating to 65°C for 30 minutes or 100°C for 10 minutes. Sialic acid is released from cell surfaces of normal and malignant cells by VCN at neutral pH without affecting viability of the cells.1, 3, 9, 20,25, 30,42 Tumor cells in M199 (pH 7.2) were incubated for 1 hour at 37°C with 25 u VCN per ml per 10 6 cells, with heat-inactivated VCN, or with M199. The cells were washed three times prior to injection as a challenging inoculum into tumor-bearing hosts. Mitomycin C Mitomycin C was obtained from Sigma Chemical Company (St. Louis, Missouri) and incubated with tumor cells in a concentration of 25 /J-g per ml per 106 cells in the same incubation mixture with VCN or heat-inactivated VCN. We have previously demonstrated that mitomycin C has no adverse effect on the efficacy of VCN-treated cells to induce immunospecific regression of firmly established methylcholanthrene fibrosarcomas in C3H/HeJ mice. Treatment of tumor cells with mitomycin C does not increase the uptake of trypan blue by such tumor cell suspensions, but effectively prevents the growth of palpable tumors from the treated cells. Transplantable Tumor Experimental Protocol The basic experimental design in transplantable tumor experiments was to inoculate adult female C3H/HeJ mice or C3HeB/FeJ mice with
MODIFIED TUMOR CELLS IN SOLID MAMMARY TUMORS
553
viable tumor cells subcutaneously into the left lateral posterior flanks. The tumors usually reached palpable size by day six and measured 0.3 to 0.9 cm by day 15. The tumors were then either left untreated or excised, and the host was challenged with VCN-treated cells into the subcutaneous tissues of the right flank. The challenging inoculum was then repeated on alternate days to a total of six inoculations. The effect ofVCNtreated tumor cells on the growth of the firmly established mammary tumors was then determined. Spontaneous Tumor Experimental Protocol Our experiments utilizing spontaneous tumors were somewhat different. Adult female C3H-HeJ mice naturally infected with the mammary tumor virus were utilized. Mice were purchased when they were 9 to 15 months old and after having delivered multiple litters at the Jackson Laboratories. The mice were examined twice weekly for the appearance of spontaneous mammary adenocarcinomas. When the tumors were found (0.7 to 0.9 cm in diameter) the mice were individually placed in precoded cages. A second investigator then utilized the code to determine the tumor treatment to be administered. Two experiments were designed. In the first, the tumor treatment consisted of intratumor injections every fourth day with 0.1 ml sterile solution containing M199, 50 u of VCN, heat-inactivated VCN, 1 mg Mycobacterium bovis (strain BCG), or a combination of VCN, heatinactivated VCN, and/or BCG. Tumor diameter was measured every 4 days just prior to next treatment, and the date of death recorded. In the second set of experiments, designed to treat spontaneous mammary tumors in retired breeding C3H/HeJ female mice, surgical excision was combined with VCN or inactivated VCN-treated autochthonous or M-I cells. When the spontaneous tumors were found, the mice were individually placed in precoded cages. Fourteen days later, when progressive tumor growth was assured, tumor excision and immunotherapy was carried out according to the cage code. The immunotherapeutic code was not revealed until after excision was performed in order to eliminate bias in the performance of the surgery. Effect of VCN-Treated Mammary Carcinoma Cells on the Growth of Established Transplantable Mammary Adenocarcinomas in Syngeneic C3H/HeJ Hosts In the left flank of recipient syngeneic C3H/HeJ mice, 40,000 M-I transplantable mammary adenocarcinoma cells were injected subcutaneously. Fifteen days thereafter, when the tumor nodules measured 0.3 to 0.8 cm in diameter, recipient mice were inoculated with 106 M-I tumor cells that had been incubated either with VCN (plus mitomycin) or heat-inactivated VCN (plus mitomycin). Results are summarized in Table 1. The injection of 10 6 M-I tumor cells exposed to heat-inactivated VCN did not affect the progressive fatal growth ofthe tumors. In contrast, injection of 106 VC N -treated M-I cells markedly slowed tumor growth and produced total regression of 4/40 firmly established M-I mammary adenocarcinomas. Animals whose tumors totally disappeared survived indefinitely, and life was significantly prolonged even in those animals
554
RICHARD
L. SIMMONS, ANGELYN Rws, AND PAUL TRITES
Table 1. Effect ofVCN-Treated M-l Mammary Adenocarcinoma Cells on Regression of M-l Tumors in C3HIHe] Mice
GROUP
TREATMENT OF CELLULAR CHALLENGE*
FRACTION OF PRIMARY TUMORS UNDERGOING TOTAL REGRESSION
MEAN TUMOR DIAMETER DAY 30 (cm ± SE)
MEAN DAY OF DEATH (± SE)**
2 3
VCN Inactive VCN
0/30 4/40 0/40
1.89 ± 0.09 1.06 ± 0.10*"* 1. 79 ± 0.07
49.4 ± 2.2 60.4 ± 3.3*** 49.3 ± 2.1
'Challenging inocula consisted of 6 injections of 106 M-I cells incubated with VCN (25 U/ml/10 6 cells) or heat-inactivated VCN (plus mitomycin C). First challenge was given 15 days after tumor inoculation and subsequent challenges at 2-d ay intervals. "*Does not include mice whose tumors totally regressed. ***p ., 0.05 when compared to groups 1 and 3 by Student's t test.
whose tumors did not totally disappear. A similar experiment was carried out with a second mammary adenocarcinoma (M-2) (Table 2). Two experiments were then done to determine the immunospecificity of the regression induced by VCN-treated mammary adenocarcinoma cells. The first such experiment is summarized in Table 3. Into the left flank of syngeneic C3H/HeJ mice, 40,000 M-I mammary adenocarcinoma cells were injected subcutaneously. Fifteen days later, tumorbearing mice were challenged with 10 6 M-I or 10 6 M-2 mammary adenocarcinoma cells that had been exposed to VCN (plus mitomycin) or heat-inactivated VCN (plus mitomycin). Slowing of tumor growth and prolongation of life in mice with M-I tumors were seen only when injections of M-I tumor exposed to VCN were given. Total regression in this series occurred in 1 of 20 animals. Tumor growth was not inhibited in recipients challenged with M-2 tumors or recipients challenged with M-I tumors treated with inactivated VCN (Table 3).
Cross-Immunization of M-I Tumors with M-2 Tumors in C3HeB/FeJ Mice These results suggested that M-I and M-2 did not share the same tumor-specific transplantation antigens. We needed to confirm this fact Table 2. Effect ofVCN-Treated M-2 Mammary Adenocarcinoma Cells on Regression of M-2 Tumors in C3HIHe] Mice
GROUP
TREATMENT OF CELLULAR CHALLENGE*
FRACTION OF PRIMARY TUMORS UNDERGOING TOTAL REGRESSION
MEAN TUMOR DIAMETER DAY 30 (cm ± SE)
MEAN DAY OF DEATH (± SE)"
1 2 3
VCN Inactive VCN
0/10 1110 0/10
1.24 ± 0.04 0.51 ± 0.08*'" 1.07 ± 0.04
78.0 ± 2.3 94.3 ± 3.7*** 72.4 ± 2.2
'Challenging inocula consisted of 6 injections of 106 M-2 cells incubated with VCN (25 U per mI per 106 cells) or heat-inactivated VCN (plus mitomycin C). First challenge was given 15 days after tumor inoculation and subsequent challenges at 2-day intervals. **Does not include mice whose tumors totally regressed. ***p ., 0.05 when compared to groups 1 and 3 by Student's t test.
555
MODIFIED TUMOR CELLS IN SOLID MAMMARY TUMORS
Table 3. Effect ofVCN-Treated M-l or M-2 Cells on Regression of M-l Tumors in C3HIHe] Mice
GROUP
CHALLENGING TUMOR
TREATMENT OF CELLULAR CHALLENGE*
FRACTION OF PRIMARY TUMORS UNDERGOING TOTAL REGRESSION
1 2 3 4 5
M-I M-I M-2 M-2
VCN Inactive VCN VCN Inactive VCN
0/20 1120 0/20 0/20 0/20
MEAN TUMOR DIAMETER, DAY 30 (cm ± SE) 1. 73 ± 1.03 ± 1.79 ± 1.81 ± 1.82 ±
0.10 0.12*** 0.10 0.08 0.09
MEAN DAY OF DEATH (± SE)** 51.0 66.8 52.7 53.5 51.4
± ± ± ± ±
3.8 4.8*** 3.3 3.2 3.2
*Challenging inocula consisted of 6 injections of M-I or M-2 cells incubated with VCN (25 U per ml per 106 cells) or heat-inactivated VCN (plus mitomycin C). First challenge was given 15 days after tumor inoculation and subsequent challenges at 2-d ay intervals. ':'*Does not include animals whose tumors totally regressed. **"p ~ 0.05 when compared to groups 1, 3, 4, and 5 by Student's t test.
before proceeding further. Therefore, 106 M-I or M-2 viable tumor cells from C3H/HeJ mice were injected subcutaneously in the left posterior flank of adult female C3HeB/FeJ mice. All injected mice developed tumors. Control mice received 106 C3H/HeJ or C3HeB/FeJ spleen cells or an equal volume (0.1 ml) of M199. Two weeks following the injection, when the tumors ranged in diameter from 0.8 to 1.6 cm, the tumor masses were totally excised, and excision of normal tissues were performed in mice not bearing tumors. Two weeks after tumor excision (one month after tumor inoculation), all mice were challenged in the subcutaneous tissues of the right posterior flank with 5x 10 3 , 5x 104 , or 5x 105 M-I orM-2 viable tumor cells. The results are recorded in Table 4. C3HeB/FeJ mice who had not received previous injections of M-I or M-2 turn or cells developed tumors from the challenging inocula which progressed to death (Table 4). C3HeB/FeJ mice pre-exposed to nonmalignant C3H/HeJ or C3HeB/FeJ spleen cells supported a similar proportion of either M-I or M-2 tumors. C3HeB/FeJ mice immunized with either M-I or M-2 tumors failed to support both M-I or M-2 tumors (P ,,; 0.05) although it seemed that mice pre-exposed to M-2 tumors supported a significantly smaller proportion of either M-I or M-2 tumors than did mice pre-exposed to M-I tumors (p ,,; 0.05). These results strongly suggest that M-I and M-2 tumors share the same mammary tumor virus tumor antigen, and that C3HeB/FeJ mice can be immunized by the mammary tumor virus antigen from the C3HI HeJ mammary tumors (Table 4). Effect of VCN-Treated Mammary Carcinoma Cells on the Growth of Simultaneous Established M-I and M·2 Tumors in Syngeneic C3H/HeJ or C3HeB/FeJ Mice 40,000 viable M-I tumor cells were injected subcutaneously into the left flank of C3H/HeJ or C3HeB/FeJ mice. Simultaneously 40,000 M-2 cells were injected in the right flank of the same mice. Thus, separate M-I and M-2 tumors were established in both the mammary tumor virus-
556
RICHARD
L.
SIMMONS, ANGELYN RIOS, AND PAUL TRITES
Table 4. Growth of M-l or M-2 Mammary Carcinomas in C3HeBIFe] Mice Pre-exposed to M-l or M-2 Tumors PRIMARY INOCULUM
None
C3H/HeJ
Spleen Cells
C3HeB/FeJ
Spleen Cells
CHALLENGING
NO. OF
FRACTION OF MICE SUPPORTING
TUMOR
CELLS
SECONDARY TUMOR
M-I
5 x 103 5 x 104 5 x 10"
3/20 14/20 20/20
M-2
5 x 103 5 x 104 5 x 10"
3/20 15/20 20/20
M-I
5 x 103 5 x 10' 5 x 10"
1/10 6/10 10/10
M-2
5 x 103 5 x 104 5 x 10"
7/10 10/10
5 x 103 5 x 104 5 x 10"
7/10 10/10
5 x 103 5 x 10' 5 x 10"
8/10 10/10
M-I
M-2
M-I
M-I
M-2
M-2
1/10
1/10
1/10
5 x 103 5 x 104 5 x 10"
2110
5 x 103 5 x 104 5 x 10"
2/10
1/10
4/10
3/10 4/10
M-I
5 x 103 5 x 104 5 x 10"
0/20
M-2
5 x 103 5 x 104 5 x 10"
0/20 0/20
1/20 1/20
1/20
infested C3H/HeJ mice and in the mammary tumor virus-free C3HeB/FeJ mice. Twelve days thereafter, after separate tumors were palpable, the mice were challenged at a third site with either 10 6 M-I or M-2 tumor cells which had been exposed to VCN (plus mitomycin) or inactivated VCN (plus mitomycin). The regression of the pre-existing tumors was then determined. The results are shown in Table 5. Inhibition of growth and regression of firmly established M-I tumors took place only in animals challenged with VCN-treated M-I tumors. M-I tumors did not regress when mice were challenged with M-I tumors exposed to inactivated VCN, nor did the M-I tumor regress when treated with M-2 cells exposed
557
MODIFIED TUMOR CELLS IN SOLID MAMMARY TUMORS
Table 5. Immunospecificity of Mammary Tumor Regression in Viral-Infested (C3H/HeJ) or Viral-Free (C3HeBIFe]) Mice Bearing Both M-l and M-2 Tumors TUMOR BEARING HOST e3H/HeJ
e3HeB/FeJ
GROUP NO.
TREATMENT OF CHALLENGING INOCULUM"
FRACTION TUMORS REGRESSING M-1 b M_2b
1 2 3 4 5
None M-I M-I M-2 M-2
M199 VeN Inactive VeN VeN Inactive VeN
0/14 3/20 0/17 0/19 0/15
0/14 0/20
6 7 8 9 10
None M-I M-I M-2 M-2
M199 VeN Inactive VeN VeN Inactive VeN
0110 6/19 0/18 0/20 0/19
MEAN TUMOR DIAMETER DAY 30 (cm ± SE) M-I M-2
0/15
0.90 0.40 0.86 1.07 1.11
± ± ± ± ±
0.07 0.05' 0.04 0.06 0.05
1.20 ± 1.32 ± 1.19 ± 0.52 ± 1.46±
0.06 0.06 0.06 0.08** 0.07
0/19 0/19 0/18 4/20 0/19
1.09 0.37 0.87 0.96 0.94
± ± ± ± ±
0.05 0.06**0.03 0.04 0.04
1.06± 1.11 ± 1.03 ± 0.42 ± 1.22 ±
0.03 0.04 0.04 0.07***0.04
0117 1119
*p ,,; 0.05 when compared to groups 1, 3, 4, 5, and contralateral tumor. **p,,; 0.05 when compared to groups 1,2, 3, 5, and contralateral tumor. ***p ,,; 0.05 when compared to groups 6, 8, 9, 10, and contralateral tumor. ****p ,,; 0.05 when compared to groups 6, 7, 8, 10, and contralateral tumor. aehallenging inocula consisted of six injections of M-I or M-2 cells which had been incubated with 25 u/ml/106 cells of veN or heat-inactivated veN (plus mitomycin e). The first challenge was given 12 days after tumor inoculation and subsequent challenges at two day intervals. bDoes not include animals whose tumors blended so that each could not be separately distinguished.
to VCN or inactivated VCN. Conversely, M-2 tumors did not regress if mice were challenged with M-2 tumors exposed to inactivated VCN, or to M-I cells in any form. The strain of the host mouse had no effect on this response. Even the C3HeB/FeJ mice free of the mammary tumor virus failed to reject M-I tumors when challenged with VCN treated M-2 cells, and vice versa. Effect of N euraminidase-Treated Mammary Adenocarcinoma Cells on the Regression of Partially and Totally Excised Adenocarcinomas in C3H/HeJ Mice 80,000 M-2 mammary adenocarcinoma cells were injected subcutaneously into the left flank of recipient C3H/HeJ mice. Fifteen or 25 days thereafter, wedge excision, enucleation, or total tumor excision was carried out and the mice were challenged with 10 6 living M-2 tumor cells which had been incubated either with VCN or heat-inactivated VCN (plus mitomycin). The results are tabulated in Table 6. Mice challenged on either day 15 or day 25 with VCN-treated tumor cells demonstrated significant slowing oftumor growth. Wedge excision of the tumor on day 15 added nothing to the immunoregressive effect of VCN-treated tumor cells. One of the 10 tumors disappeared even without tumor excision. When treatment was delayed until day 25, tumor re growth was significantly inhibited by a combination of partial, subtotal, or attempted
Total Total
25 25
VCN Inactive VCN
7/10 3/10
1110 0110
0/10 0/10 0/10
0/10 0/10
0/10 1110 0/10
REGRESSING
0.61 ± 0.05 0.63 ± 0.06 0.00 ± 0.00 0.00 ± 0.00
0.91 ± 0.03 0.92 ± 0.03
1.19 ± 0.06 1.08 ± 0.06 1.11 ± 0.04
0.96 ± 0.05 0.95 ± 0.04 0.91 ± 0.04 0.95 ± 0.05 0.90 ± 0.03
0.27 ± 0.02 0.30 ± 0.02
0.29 ± 0.03 0.30 ± 0.03 0.31 ± 0.03
24 hr post excision
0.25 ± 0.02 0.28 ± 0.03
0.27 ± 0.03 0.26 ± 0.03 0.28 ± 0.03
24 hr prior to excision
SE)
2 weeks post excision
±
0.14 ± 0.07"'* 0.33 ± 0.16**
0.98 ± 0.13**t 2.22 ± 0.18
2.61 ± 0.19 1.85 ± 0.17 2.78 ± 0.09
0.86 ± 0.15* 1.03 ± 0.21
1.47 ± 0.09 0.56 ± 0.10* 1.44 ± 0.08
MEAN TUMORDIAMETER (cm
SE)b
103.7 ± 13.0**t 77.6 ± 13.5*'"
87.0 ± 4.4**t 65.0 ± 3.5
60.9 ± 4.9 74.7 ± 3.1**t 59.8 ± 3.0
61.0±5.7 59.7 ± 5.1
60.9 ± 4.9 65.9 ± 5.9 64.0 ± 4.2
(±
OF DEATH
MEAN DAY
6
aChallenging inocula consisted of six injections of 10 M-2 cells, the first challenge was given 15 or 25 days after tumor inoculation with subsequent challenges at 2 day intervals. bDoes not include mice whose tumors totally regressed. *p ,;; 0.05 when compared to groups 1, 3, and 5 (Student's T test). "'*p ,;; 0.05 when compared to groups 6, 8, and 10 (Student's T test). tp ,;; 0.05 when compared with mice challenged with inactive VCN treated cells (Student's T test). ttGroups 1 and 6 refer to the same animals, tumors being measured at differing intervals after implantation.
12
11
VCN Inactive VCN
Enucleation Enucleation
25 25
9 10
VCN Inactive VCN
None None None
25 25
6tt 7 8
VCN Inactive VCN
Wedge Wedge
15 15
4 5
VCN Inactive VCN
15 15
Ht 2 3
None None None
(DAYS)
NO.
CHALLENGE a
CELLULAR
TREATMENT
GROUP
EXCISION
PRIMARY TUMORS
TREATMENT OF
IMPLANTATION AND TOTALLY
FRACTION OF
INTERVAL BETWEEN
Table 6. Effect of Tumor Excision and Immunotherapy on the Growth of M-2 Tumors in C3HIHej Mice
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559
MODIFIED TUMOR CELLS IN SOLID MA~MARY TUMORS
total excision plus challenge with VCN-treated tumor cells. Seven of 10 animals were cured by at~empted total tumor excision plus VCN-treated tumor cells. Partial tumor excision plus challenge with heat-inactivated VCN -treated tumor cells did not significantly inhibit tumor regrowth, and total excision plus inaetivated VCN-treated, tumorcells cured on\y three of 10 animals. Effect of Direct In.~ra~umor Injections of Neuraminidase ancJ/or BCG into Spontaneous Mammary Adenocarcinomas in C3H/HeJ Mice It is possible th:1lit transplantable mammary tumors do not resemble spontaneous mammary tumors in their responses to immunotherapy with neuraminidase-treated cells. Since we had shown that the repeated intralesional injection of-VGN into transplantable fibrosarcomas le
