Effect of Corynebacterium parvum on Tumor Growth in Normal and Athymic (Nude) Mice 1,2,3 Michael F. A. Woodruff 4 and Noel L. Warners,s ABSTRACT-The effect of systemic or local Injection of Coryne-
bacterium parvum at the tumor site on the growth of various murine tumors was studied in Intact and congenitally athymic BALB/c mice. Systemic injection of C. parvum usually had a marked antitumor effect in both types of mouse. Two lymphomas, which regressed spontaneously In untreated intact mice but not In athymlc mice, grew progressively In Intact mice given systemic C. parvum, though their growth was Inhibited In similarly treated athymic mice. Local injection into the site of the tumor markedly inhibited tumor growth in intact mice but was without effect in athymic mice. C. parvum was believed to exert its antitumor effects by two different mechanisms, only one of which was T-cell dependent. The mechanism not dependent on T-cells was particularly activated by systemic C. parvum Injection.-J Natl Cancer Inst 58: 111-116, 1977.
Systemic injection (iv or ip) of a killed suspension of an active strain of Corynebacterium parvum appears to inhibit the growth of subcutaneous isotransplants of cholanthrene-induced murine sarcomas as effectively in mice made T-cell deficient by thymectomy, whole-body irradiation, and isogenic bone marrow transplantation, as in intact mice (1). Injection ofC. parvum at the site of tumor inoculation, however, though highly effective in intact mice (2-4), has little or no effect on the growth of isotransplants of a mastocytoma (3) and a cholanthreneinduced sarcoma (4) in T -cell-deficient mice. Prior exposure to 500-rads of whole-body irradiation, which may reduce but not eliminate the systemic effect of C. parvum on phagocytic activity in adult CBA mice, reduced but did not abolish the antitumor effect of a systemic (ip) injection of C. parvum in this strain, whereas it completely abrogated the effect of an intratumor injection (5). These findings and the observation that peritoneal macrophages from both intact and Tcell-deficient mice inoculated with C. parvum are highly cytotoxic for tumor cells in vitro (6, 7) suggest that the antitumor effect of C. parvum may be mediated in two ways. The first mechanism, which appears to predominate when C. parvum is given systemically, is independent of T-cells and requires widespread activation of macrophages. The second involves T-cells and is not dependent on widespread macrophage stimulation, though macrophage activation may be involved in the immediate vicinity of the tumor. Because other observations on adjuvant activity of C. parvum suggest possible discordance between results in athymic versus T-cell-depleted mice (8, 9), we have further investigated the antitumor effect of C. parvum on syngeneic transplants of a variety of tumors in normal and congenitally athymic (nu/nu) mice.
place of inbred BALB/c mice, because the tumors grew equally well in the inbred or hybrid mice. The athymic (BALB/c nu/nu) mice, which were specific-pathogenfree, were in backcross generations 5-7. The colony prior to this stage had been selected for H-2d homozygotes, and control studies have shown that various BALB/c-derived tumors grow equally well in the BALB/c inbred mice and BALB/c nu/+ heterozygotes. All mice were raised and maintained at the Hall Institute under the supervision of Dr. M. Holmes. Tumors . -The tumor lines used in this study are listed in table I with their mode of maintenance and type. All tumors were of inbred BALB/c origin. Tumor lines, maintained in tissue culture as continuous stationary suspension cultures as previously described (10), were provided by Drs. Harris and Burton. 5 All the tumors used were previously shown by either in vivo or in vitro techniques to express tumor-specific transplantation antigens (11-13). More extensive studies of the growth characteristics of these tumors in untreated athymic BALB/c mice will be described elsewhere. 7 Tumor cell suspensions were prepared in Eisen's balanced salt solution with 10% fetal calf serum, and the number of viable cells was determined by eosin dye exclusion. The required dose of cells was contained in 0.1 ml and inoculated sc in the right flanks. The mice were examined every 2-3 days and the mean tumor diameter was recorded. All experiments involved groups of 6-8 mice. At the end of each experiment, mice were killed by neck dislocation and tumor tissue was dissected and weighed. C. parvum.-A formol-killed suspension of C. parvum strain CN6134 (batch PX425; Wellcome Foundation) was used throughout. The original suspension containing 7 mg (dry wt) organisms/ml was used undiluted in a dosage of 0.1 ml or 0.2 ml for sc injection at the site of tumor inoculation (intratumor) or elsewhere (sc). For iv injection, the suspension was usually diluted with an equal volume of normal saline, but occasionally undiAbbreviations used: GVH = graft versus host; MLC = mixed leukocyte; PHA = phytohemagglutinin. Received February 18, 1976; accepted May 17, 1976. Supported by Public Health Service contract NIH-NCI-G-72-3889 from the National Cancer Institute. 3 Publication No. 2147 from The Walter and Eliza Hall Institute of Medical Research. 4 Department of Surgery, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland. 5 Genetics Unit, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. 6 We are grateful to Dr. A. N. Weinberg, Wellcome Foundation Research Laboratories, Beckenham, Kent, England, for providing the C. parvum. One of us (M.F.A.W.) is deeply indebted to his coauthor and to the Director and staff of the Hall Institute for their generous provision of facilities and an intellectually stimulating environment during his sabbatical leave. 7 Warner NL, Woodruff MF: In preparation. 1
2
MATERIALS AND METHODS
Mice.-Normal and congenitally athymic (nude) male and female BALB/c mice between 6 and 12 weeks old were used in most of the experiments. Occasionally, as noted, (BALB/c X NZB)Fl (BNF 1) mice were used in VOL. 58, NO.1, JANUARY 1977
III
J NATL CANCER INST
112
WOODRUFF AND WARNER TABLE
Source (reference)
Strain of origin
Tumor type
Hall Institute (10) M. Scharff, New York U2) Hall Institute (9) Hall Institute (13) M. Potter U3) M. Potter Hall Institute (13) Hall Institute
BALB/c BALB/c BALB/c BALB/c BALB/c BALB/c BNFI BALB/c
Fibrosarcoma Plasma cell tumor Lymphoma (T-cell) Lymphoma (B-cell) Lymphoma (B-cell) Lymphoma (B-cell) Plasma cell tumor Carcinoma
Designation WEHI164
MPcn WEHI 22 WEHI212 ABEl ABE8 HPC-158 WEHI 100
I.-Tumors studied Maintenence Tissue culture Tissue culture Tissue culture Serial transplantation Serial transplantation Serial transplantation Serial transplantation Serial transplantation
10
luted suspension was used; in either case, the volume injected was 0.2 ml. All injections were given 3 days after tumor inoculation. RESULTS
The effect of C. parvum administration by various routes on the subcutaneous growth of various transplantable tumors was assessed in both syngeneic normal and athymic mice. The detailed results obtained with each tumor are presented. Group mean tumor diameters with bars to indicate the standard error are plotted in the text-figures.
E E
I.-Growth of fibrosarcoma WEHI-I64 in BALB/c normal mice. o--o=controI, no treatment; Ji.----Ji.=C. parvum (0.7 mg) given iv; e----e=C. parvum (0.7 mg) injected into tumor site. All mice received 7 X 10' tumor cells.
TEXT-FIGURE
Ck:
w w 5 ~
>-
/
-
,
, ,,
Plasmacytoma MPC-11
Intratumor injection of MPC-11 with C. parvum caused significant tumor growth inhibition in normal mice (text-fig. 3A), whereas little or no inhibition (textfig. 3B) occurred in athymic mice. Tumor MPC-ll grew only poorly in control athymic mice; this difference in tumor growth rate in normal and athymic mice is the subject of a separate communication. 7 Determination of tumor weights in both treated and control athymic and normal mice also showed the inhibitory effect of C. parvum only in the normal treated mice (table 2); a similar degree of tumor inhibition was observed in normal mice given C. parvum iv or by injection into the tumor. The sc injection of C. parvum in normal mice at a site remote from tumor cell inoculation was completely ineffective in both normal (text-fig. 3A) and athymic mice.
J
NATL CANCER INST
/1
'f-- ~-1
,,' ... ,-,
~ I'
5
Fibrosarcoma WEHI-164
The iv injection of 1.4 mg C. parvum 3 days after tumor injection markedly inhibited growth of the tumor in normal mice. A lesser, but still inhibitory, effect was observed with 0.7 mg injected iv 3 days after tumor inoculation (text-fig. 1). Injection into the site of the tumor was given 3 days after tumor inoculation; it also clearly inhibited growth rate (text-fig. 1). Mean tumor weights (mg±sE) taken at day 17 similarly showed C. parvum inhibitory effects (table 2). In contrast to the significant inhibition observed in tumor growth in normal mice receiving intratumor injections of C. parvum, tumor growth in athymic mice was not inhibited by this treatment (text-fig. 2). However, in both experiments iv injection of C. parvum in the athymic mice inhibited tumor growth.
(solid tumor) (ascites) (ascites) (solid tumor) (solid tumor)
10
15
DAYS AFTER INOCULATION B-Lymphomas ABE-1 and ABE-8
C. parvum injected iv caused slight inhibition of the growth of ABE-8 in both normal and athymic mice. Injection of C. parvum into the site of tumor inoculation markedly inhibited the growth of this tumor in normal mice (text-fig. 4A) but did not affect its growth in athymic mice (text-fig. 4B). ABE-I was studied in athymic mice only. Once again, growth was inhibited by iv injection of C. parvum but not by tumor-site injection (text-fig. 5). Lack of Susceptibility of Some Tumors to C. parvum Inhibition
The studies described in the preceding sections showed C. parvum-mediated inhibition of 4 different transplantable tumors. However, we have observed that some murine tumor lines may not be susceptible to C. parvum-mediated inhibition. The results in text-figure 6 show failure to inhibit growth of plasmacytoma HPC 158 and carcinoma WEHI-lOO in normal mice by intratumor or iv injection, respectively. C. parvum injected iv resulted in a growth curve for HPCI58 which coincided with that following intratumor injection (mean diameter at 12 days, 8.67±0.6I mm after iv and 8.83±0.48 mm after intratumor injection). In these studies the dose of tumor cells was such that the control growth rate was comparable to that of tumors susceptible to C. parvum. Effect of C. parvum on Growth of Lymphomas That Undergo Spontaneous Regression
The transplantation of several lymphomas in normal syngeneic mice was followed by spontaneous regression
VOL. 58, NO.1, JANUARY 1977
113
EFFECT OF C. PARVUM ON TUMORS IN NORMAL AND NUDE MICE
TABLE 2.-Effect of C. parvum on subcutaneous tumor growth
a
Tumor weight, in mg, at autopsy b Number of viable cells inoculated
Tumor
Recipients
7x105 9.6x105 1.2 x 10· 1.2x10·
WEHI-164 MPC-ll MPC-ll MPC-ll
N N N nu
Duration of experiment days
After injection of C. parvum on day 3 Untreated iv
17 19 21 21
Intratumor 171±55 326±1l1 288±139 679±379
243±63 604±104
379±53 1,405±214 5,150±600 523±160
a Groups of intact (N) or athymic (nu) mice were given injections oftumor sc and 3 days later were given either C. parvum iv, at the tumor site, or no injection. Tumors were dissected at the indicated time and weighed. b Values are meanS±SE.
10
10 A
E E
B
20
A
20
0::: UJ fUJ
::E
bacterium parvum at the tumor site on the growth of various murine tumors was studied in Intact and congenitally athymic BALB/c mice. Systemic injection of C. parvum usually had a marked antitumor effect in both types of mouse. Two lymphomas, which regressed spontaneously In untreated intact mice but not In athymlc mice, grew progressively In Intact mice given systemic C. parvum, though their growth was Inhibited In similarly treated athymic mice. Local injection into the site of the tumor markedly inhibited tumor growth in intact mice but was without effect in athymic mice. C. parvum was believed to exert its antitumor effects by two different mechanisms, only one of which was T-cell dependent. The mechanism not dependent on T-cells was particularly activated by systemic C. parvum Injection.-J Natl Cancer Inst 58: 111-116, 1977.
Systemic injection (iv or ip) of a killed suspension of an active strain of Corynebacterium parvum appears to inhibit the growth of subcutaneous isotransplants of cholanthrene-induced murine sarcomas as effectively in mice made T-cell deficient by thymectomy, whole-body irradiation, and isogenic bone marrow transplantation, as in intact mice (1). Injection ofC. parvum at the site of tumor inoculation, however, though highly effective in intact mice (2-4), has little or no effect on the growth of isotransplants of a mastocytoma (3) and a cholanthreneinduced sarcoma (4) in T -cell-deficient mice. Prior exposure to 500-rads of whole-body irradiation, which may reduce but not eliminate the systemic effect of C. parvum on phagocytic activity in adult CBA mice, reduced but did not abolish the antitumor effect of a systemic (ip) injection of C. parvum in this strain, whereas it completely abrogated the effect of an intratumor injection (5). These findings and the observation that peritoneal macrophages from both intact and Tcell-deficient mice inoculated with C. parvum are highly cytotoxic for tumor cells in vitro (6, 7) suggest that the antitumor effect of C. parvum may be mediated in two ways. The first mechanism, which appears to predominate when C. parvum is given systemically, is independent of T-cells and requires widespread activation of macrophages. The second involves T-cells and is not dependent on widespread macrophage stimulation, though macrophage activation may be involved in the immediate vicinity of the tumor. Because other observations on adjuvant activity of C. parvum suggest possible discordance between results in athymic versus T-cell-depleted mice (8, 9), we have further investigated the antitumor effect of C. parvum on syngeneic transplants of a variety of tumors in normal and congenitally athymic (nu/nu) mice.
place of inbred BALB/c mice, because the tumors grew equally well in the inbred or hybrid mice. The athymic (BALB/c nu/nu) mice, which were specific-pathogenfree, were in backcross generations 5-7. The colony prior to this stage had been selected for H-2d homozygotes, and control studies have shown that various BALB/c-derived tumors grow equally well in the BALB/c inbred mice and BALB/c nu/+ heterozygotes. All mice were raised and maintained at the Hall Institute under the supervision of Dr. M. Holmes. Tumors . -The tumor lines used in this study are listed in table I with their mode of maintenance and type. All tumors were of inbred BALB/c origin. Tumor lines, maintained in tissue culture as continuous stationary suspension cultures as previously described (10), were provided by Drs. Harris and Burton. 5 All the tumors used were previously shown by either in vivo or in vitro techniques to express tumor-specific transplantation antigens (11-13). More extensive studies of the growth characteristics of these tumors in untreated athymic BALB/c mice will be described elsewhere. 7 Tumor cell suspensions were prepared in Eisen's balanced salt solution with 10% fetal calf serum, and the number of viable cells was determined by eosin dye exclusion. The required dose of cells was contained in 0.1 ml and inoculated sc in the right flanks. The mice were examined every 2-3 days and the mean tumor diameter was recorded. All experiments involved groups of 6-8 mice. At the end of each experiment, mice were killed by neck dislocation and tumor tissue was dissected and weighed. C. parvum.-A formol-killed suspension of C. parvum strain CN6134 (batch PX425; Wellcome Foundation) was used throughout. The original suspension containing 7 mg (dry wt) organisms/ml was used undiluted in a dosage of 0.1 ml or 0.2 ml for sc injection at the site of tumor inoculation (intratumor) or elsewhere (sc). For iv injection, the suspension was usually diluted with an equal volume of normal saline, but occasionally undiAbbreviations used: GVH = graft versus host; MLC = mixed leukocyte; PHA = phytohemagglutinin. Received February 18, 1976; accepted May 17, 1976. Supported by Public Health Service contract NIH-NCI-G-72-3889 from the National Cancer Institute. 3 Publication No. 2147 from The Walter and Eliza Hall Institute of Medical Research. 4 Department of Surgery, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland. 5 Genetics Unit, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. 6 We are grateful to Dr. A. N. Weinberg, Wellcome Foundation Research Laboratories, Beckenham, Kent, England, for providing the C. parvum. One of us (M.F.A.W.) is deeply indebted to his coauthor and to the Director and staff of the Hall Institute for their generous provision of facilities and an intellectually stimulating environment during his sabbatical leave. 7 Warner NL, Woodruff MF: In preparation. 1
2
MATERIALS AND METHODS
Mice.-Normal and congenitally athymic (nude) male and female BALB/c mice between 6 and 12 weeks old were used in most of the experiments. Occasionally, as noted, (BALB/c X NZB)Fl (BNF 1) mice were used in VOL. 58, NO.1, JANUARY 1977
III
J NATL CANCER INST
112
WOODRUFF AND WARNER TABLE
Source (reference)
Strain of origin
Tumor type
Hall Institute (10) M. Scharff, New York U2) Hall Institute (9) Hall Institute (13) M. Potter U3) M. Potter Hall Institute (13) Hall Institute
BALB/c BALB/c BALB/c BALB/c BALB/c BALB/c BNFI BALB/c
Fibrosarcoma Plasma cell tumor Lymphoma (T-cell) Lymphoma (B-cell) Lymphoma (B-cell) Lymphoma (B-cell) Plasma cell tumor Carcinoma
Designation WEHI164
MPcn WEHI 22 WEHI212 ABEl ABE8 HPC-158 WEHI 100
I.-Tumors studied Maintenence Tissue culture Tissue culture Tissue culture Serial transplantation Serial transplantation Serial transplantation Serial transplantation Serial transplantation
10
luted suspension was used; in either case, the volume injected was 0.2 ml. All injections were given 3 days after tumor inoculation. RESULTS
The effect of C. parvum administration by various routes on the subcutaneous growth of various transplantable tumors was assessed in both syngeneic normal and athymic mice. The detailed results obtained with each tumor are presented. Group mean tumor diameters with bars to indicate the standard error are plotted in the text-figures.
E E
I.-Growth of fibrosarcoma WEHI-I64 in BALB/c normal mice. o--o=controI, no treatment; Ji.----Ji.=C. parvum (0.7 mg) given iv; e----e=C. parvum (0.7 mg) injected into tumor site. All mice received 7 X 10' tumor cells.
TEXT-FIGURE
Ck:
w w 5 ~
>-
/
-
,
, ,,
Plasmacytoma MPC-11
Intratumor injection of MPC-11 with C. parvum caused significant tumor growth inhibition in normal mice (text-fig. 3A), whereas little or no inhibition (textfig. 3B) occurred in athymic mice. Tumor MPC-ll grew only poorly in control athymic mice; this difference in tumor growth rate in normal and athymic mice is the subject of a separate communication. 7 Determination of tumor weights in both treated and control athymic and normal mice also showed the inhibitory effect of C. parvum only in the normal treated mice (table 2); a similar degree of tumor inhibition was observed in normal mice given C. parvum iv or by injection into the tumor. The sc injection of C. parvum in normal mice at a site remote from tumor cell inoculation was completely ineffective in both normal (text-fig. 3A) and athymic mice.
J
NATL CANCER INST
/1
'f-- ~-1
,,' ... ,-,
~ I'
5
Fibrosarcoma WEHI-164
The iv injection of 1.4 mg C. parvum 3 days after tumor injection markedly inhibited growth of the tumor in normal mice. A lesser, but still inhibitory, effect was observed with 0.7 mg injected iv 3 days after tumor inoculation (text-fig. 1). Injection into the site of the tumor was given 3 days after tumor inoculation; it also clearly inhibited growth rate (text-fig. 1). Mean tumor weights (mg±sE) taken at day 17 similarly showed C. parvum inhibitory effects (table 2). In contrast to the significant inhibition observed in tumor growth in normal mice receiving intratumor injections of C. parvum, tumor growth in athymic mice was not inhibited by this treatment (text-fig. 2). However, in both experiments iv injection of C. parvum in the athymic mice inhibited tumor growth.
(solid tumor) (ascites) (ascites) (solid tumor) (solid tumor)
10
15
DAYS AFTER INOCULATION B-Lymphomas ABE-1 and ABE-8
C. parvum injected iv caused slight inhibition of the growth of ABE-8 in both normal and athymic mice. Injection of C. parvum into the site of tumor inoculation markedly inhibited the growth of this tumor in normal mice (text-fig. 4A) but did not affect its growth in athymic mice (text-fig. 4B). ABE-I was studied in athymic mice only. Once again, growth was inhibited by iv injection of C. parvum but not by tumor-site injection (text-fig. 5). Lack of Susceptibility of Some Tumors to C. parvum Inhibition
The studies described in the preceding sections showed C. parvum-mediated inhibition of 4 different transplantable tumors. However, we have observed that some murine tumor lines may not be susceptible to C. parvum-mediated inhibition. The results in text-figure 6 show failure to inhibit growth of plasmacytoma HPC 158 and carcinoma WEHI-lOO in normal mice by intratumor or iv injection, respectively. C. parvum injected iv resulted in a growth curve for HPCI58 which coincided with that following intratumor injection (mean diameter at 12 days, 8.67±0.6I mm after iv and 8.83±0.48 mm after intratumor injection). In these studies the dose of tumor cells was such that the control growth rate was comparable to that of tumors susceptible to C. parvum. Effect of C. parvum on Growth of Lymphomas That Undergo Spontaneous Regression
The transplantation of several lymphomas in normal syngeneic mice was followed by spontaneous regression
VOL. 58, NO.1, JANUARY 1977
113
EFFECT OF C. PARVUM ON TUMORS IN NORMAL AND NUDE MICE
TABLE 2.-Effect of C. parvum on subcutaneous tumor growth
a
Tumor weight, in mg, at autopsy b Number of viable cells inoculated
Tumor
Recipients
7x105 9.6x105 1.2 x 10· 1.2x10·
WEHI-164 MPC-ll MPC-ll MPC-ll
N N N nu
Duration of experiment days
After injection of C. parvum on day 3 Untreated iv
17 19 21 21
Intratumor 171±55 326±1l1 288±139 679±379
243±63 604±104
379±53 1,405±214 5,150±600 523±160
a Groups of intact (N) or athymic (nu) mice were given injections oftumor sc and 3 days later were given either C. parvum iv, at the tumor site, or no injection. Tumors were dissected at the indicated time and weighed. b Values are meanS±SE.
10
10 A
E E
B
20
A
20
0::: UJ fUJ
::E
