Vol. 23, No. 1
INFECTION AND IMMUNITY, Jan. 1979, p. 54-60 0019-9567/79/01-0054/07$02.00/0
Antitumor Activity of Listeria monocytogenes on a Guinea Pig Fibrosarcoma MEHER M. DUSTOOR,'t AMY FULTON,2 WILLIAM CROFT,2 AND ANDREW A. BLAZKOVEC'* Departments of Medical Microbiology' and Human Oncology,2 University of Wisconsin-Madison, Madison, Wisconsin 53706 Received for publication 26 October 1978
Listeria monocytogenes-mediated tumor inhibition was studied in strain 13 guinea pigs by using a methylcholanthrene-induced fibrosarcoma (MCA-1). Mixtures of Listeria and tumor cells in ratios of 1:100, 1:200, or 1:400 (Listeria:MCA1 cells) led to significant suppression of tumor growth. Intralesional injection of tumors on day 6 posttransplantation led to the regression of a highly significant number of tumors. Animals receiving injections of Listeria, either in a mixture with tumor cells or intralesionally, displayed enhanced skin test reactivity to a tumor extract. Tumor regressors were resistant for at least 2 to 3 months after the initial transplant to rechallenge with MCA-1 cells. Thus, with this particular tumor-host system, Listeria was successfully employed as an antitumor agent with no visibly detrimental side effects to the host. Microorganisms which have been used for clinical and experimental immunotherapy of tumors included BCG (2, 14,30), Corynebacterium parvum (11, 21), Brucella abortus (26), Bordetella pertussis (15, 24), Toxoplasma gondii (13), and Listeria monocytogenes (3, 28, 29). In addition to being potent reticuloendothelial system stimulants, many of these agents evoke an acquired cellular resistance in the host, which to a great measure nonspecifically inhibits tumor growth (10). In a number of animal species, acquired cellular resistance plays a major role in recovery from a Listeria infection (9, 16, 18). A number of reports in the literature have also documented the adjuvant and mitogenic properties of Listeria extracts (6, 7) as well as of whole cells (12). Studies with two separate murine fibrosarcoma systems have shown the successful use of L. monocytogenes in tumor inhibition (3, 28). With a guinea pig hepatoma, the use of Listeria as a therapeutic agent produced equivocal results (4). Thus, it was deemed of interest to study the antitumor activity of Listeria by utilizing a different tumor-host system. The studies reported here were performed in inbred strain 13 guinea pigs with a transplantable methylcholanthreneinduced fibrosarcoma. (This work was performed by M.M.D. in partial fulfillment of the requirements for a Ph.D. degree from the University of Wisconsin, Madison).
MATERIALS AND METHODS Animals. Inbred strain 13 guinea pigs of both sexes were obtained from the breeding colony maintained in the animal care facilities at the University of Wisconsin, Madison. Tumor. The tumor designated MCA-1 was induced by the subcutaneous injection of 20 mg of 3-methylcholanthrene in 0.25 ml of corn oil into a strain 13 guinea pig. Histologically it was characterized as a fibrosarcoma and consisted of pleomorphic, spindleshaped cells. The tumor was maintained by serial intraperitoneal transplantation. To ensure the use of tumor cells from the same transplant generation throughout this study, a large stock of tumor cells from the transplant generation 18 was prepared and stored at -196°C in liquid nitrogen. Bacteria. The strain of L. monocytogenes used and the method of cultivation have been described elsewhere (8). In most of the experiments cited, viable organisms were utilized. Organisms were killed by heating in a boiling water bath for 15 min. Preparation of single-cell suspensions of tumor. For any given experiment, a single animal bearing a palpable tumor served as a source of tumor cells. The tumor mass was aseptically removed, freed of all necrotic and hemorrhagic areas, and finely minced before trypsinization (0.25% trypsin; Difco Laboratories, Detroit, Mich.) in phosphate-buffered saline at 37°C for 25 min. This was followed by filtration through gauze and the addition of sufficient fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.) to achieve a final concentration of 10%. After centrifugation at 150 x g for 8 min, cell viability was estimated by the trypan blue exclusion test. The cells were again centrifuged and suspended to the desired concentration in medium 199 (Grand Island Biological Co.). Injection of tumor cells and assessment of tu-
t Present address: Department of Pediatrics, University of Wisconsin Medical Center, Madison, WI 53706.
54
VOL. 23, 1979
TUMOR INHIBITION WITH LISTERIA IN GUINEA PIGS
mor growth. The desired number of tumor cells suspended in 0.2 ml of medium 199 with or without Listeria was injected subcutaneously in a shaved abdominal area with a 20-gauge, 1-inch (ca. 2.5-cm) needle. Intratumoral injections of Listeria were performed by using a volume of 0.2 ml of saline and a 26-gauge needle. The needle was inserted into the tumor mass with the bevel up, and the entire tumor was irrigated by rotating the needle in a 1800 arc. The aniimals were monitored for tumor growth at regular intervals over a 3-month period. The size of the tumor is expressed as the mean tumor diameter computed after measuring two perpendicular tumor diameters. Preparation of tumor antigen. A 3 M KCI extract of the tumor was prepared by the method of Meltzer et al. (19) by using 2 ml of 3 M KCI in 0.005 M potassium phosphate buffer (pH 7.2) per g of tumor. The antigen solution thus obtained was assayed for protein concentration by the biuret test, distributed into vials, and lyophilized. Measurements and evaluation of skin reactions. Skin reactions were measured by using the following three criteria: (i) degree of erythema, (ii) mean diameter of reaction, and (iii) changes in the double thickness of skin at the site of reaction. Double skin thickness was evaluated with a Schnelltaster as described previously (5). These three criteria were arbitrarily evaluated as shown in Table 1.
55
percent mortality and also gave the most uniform pattern of growth. The tumor grew as a localized subcutaneous mass which ulcerated through the skin. Penetration of the peritoneal cavity was a very rare occurrence. Lung metastases were present at autopsy in about 75% of the animals. Effect of Listeria on the growth of MCA1 cells. (i) Listeria inoculated in mixture with tumor cells. Listeria cells were injected with 2.5 x 106 MCA-1 cells into strain 13 guinea pigs in ratios of Listeria to tumor cells of 1:100, 1:200, and 1:400. Animals injected with tumor cells alone served as controls. Figures 1 through TABLE 2. Incidence of mortality in guinea pigs injected with different doses of MCA-1 cells Tumor cell No. dead/no. Survival timea dose injected (days) 1 x 107 6/10 38, 45, 45, 48, 55, 86 5 x 106 7/10 24, 41, 54, 61, 79, 83, 106 2.5 x 106 8/10 61, 61, 61, 65, 66, 75, 79, 79 1 x 106 5/10 49,61,75,75,75 5 x 10 3/9 59, 59, 89 a Animals were kept under observation for 120 days. The remaining animals in each group were alive and tumor-free at the end of the observation period. 45.0
RESULTS Growth characteristics of different subcutaneous doses of MCA-1 tumor cells. To determine the lowest dose of MCA-1 cells which would lead to progressive tumor growth in strain 13 guinea pigs, groups of 10 sex-matched animals were injected subcutaneously with 1 x 107, 5 x 106, 2.5 x 106, 1 x 106, or 5 x 105 MCA-1 tumor cells. Table 2 shows the survival times of the progressor. Up to a certain point the mortality rate was dose dependent. With doses above 2.5 x 106 cells, however, there was no marked increase in mortality. The dose of 2.5 x 106 cells was therefore chosen for future experiments because it was the lowest dose which resulted in the highest
40.0 35.0-
2-30.0* 25.0-
° 20.0
{l
Z 15.0 La
5.0
TABLE 1. Evaluation of skin reactions' Score
Erythema
Diameter (mm)
Double skin thickness (mm x 10)
INFECTION AND IMMUNITY, Jan. 1979, p. 54-60 0019-9567/79/01-0054/07$02.00/0
Antitumor Activity of Listeria monocytogenes on a Guinea Pig Fibrosarcoma MEHER M. DUSTOOR,'t AMY FULTON,2 WILLIAM CROFT,2 AND ANDREW A. BLAZKOVEC'* Departments of Medical Microbiology' and Human Oncology,2 University of Wisconsin-Madison, Madison, Wisconsin 53706 Received for publication 26 October 1978
Listeria monocytogenes-mediated tumor inhibition was studied in strain 13 guinea pigs by using a methylcholanthrene-induced fibrosarcoma (MCA-1). Mixtures of Listeria and tumor cells in ratios of 1:100, 1:200, or 1:400 (Listeria:MCA1 cells) led to significant suppression of tumor growth. Intralesional injection of tumors on day 6 posttransplantation led to the regression of a highly significant number of tumors. Animals receiving injections of Listeria, either in a mixture with tumor cells or intralesionally, displayed enhanced skin test reactivity to a tumor extract. Tumor regressors were resistant for at least 2 to 3 months after the initial transplant to rechallenge with MCA-1 cells. Thus, with this particular tumor-host system, Listeria was successfully employed as an antitumor agent with no visibly detrimental side effects to the host. Microorganisms which have been used for clinical and experimental immunotherapy of tumors included BCG (2, 14,30), Corynebacterium parvum (11, 21), Brucella abortus (26), Bordetella pertussis (15, 24), Toxoplasma gondii (13), and Listeria monocytogenes (3, 28, 29). In addition to being potent reticuloendothelial system stimulants, many of these agents evoke an acquired cellular resistance in the host, which to a great measure nonspecifically inhibits tumor growth (10). In a number of animal species, acquired cellular resistance plays a major role in recovery from a Listeria infection (9, 16, 18). A number of reports in the literature have also documented the adjuvant and mitogenic properties of Listeria extracts (6, 7) as well as of whole cells (12). Studies with two separate murine fibrosarcoma systems have shown the successful use of L. monocytogenes in tumor inhibition (3, 28). With a guinea pig hepatoma, the use of Listeria as a therapeutic agent produced equivocal results (4). Thus, it was deemed of interest to study the antitumor activity of Listeria by utilizing a different tumor-host system. The studies reported here were performed in inbred strain 13 guinea pigs with a transplantable methylcholanthreneinduced fibrosarcoma. (This work was performed by M.M.D. in partial fulfillment of the requirements for a Ph.D. degree from the University of Wisconsin, Madison).
MATERIALS AND METHODS Animals. Inbred strain 13 guinea pigs of both sexes were obtained from the breeding colony maintained in the animal care facilities at the University of Wisconsin, Madison. Tumor. The tumor designated MCA-1 was induced by the subcutaneous injection of 20 mg of 3-methylcholanthrene in 0.25 ml of corn oil into a strain 13 guinea pig. Histologically it was characterized as a fibrosarcoma and consisted of pleomorphic, spindleshaped cells. The tumor was maintained by serial intraperitoneal transplantation. To ensure the use of tumor cells from the same transplant generation throughout this study, a large stock of tumor cells from the transplant generation 18 was prepared and stored at -196°C in liquid nitrogen. Bacteria. The strain of L. monocytogenes used and the method of cultivation have been described elsewhere (8). In most of the experiments cited, viable organisms were utilized. Organisms were killed by heating in a boiling water bath for 15 min. Preparation of single-cell suspensions of tumor. For any given experiment, a single animal bearing a palpable tumor served as a source of tumor cells. The tumor mass was aseptically removed, freed of all necrotic and hemorrhagic areas, and finely minced before trypsinization (0.25% trypsin; Difco Laboratories, Detroit, Mich.) in phosphate-buffered saline at 37°C for 25 min. This was followed by filtration through gauze and the addition of sufficient fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.) to achieve a final concentration of 10%. After centrifugation at 150 x g for 8 min, cell viability was estimated by the trypan blue exclusion test. The cells were again centrifuged and suspended to the desired concentration in medium 199 (Grand Island Biological Co.). Injection of tumor cells and assessment of tu-
t Present address: Department of Pediatrics, University of Wisconsin Medical Center, Madison, WI 53706.
54
VOL. 23, 1979
TUMOR INHIBITION WITH LISTERIA IN GUINEA PIGS
mor growth. The desired number of tumor cells suspended in 0.2 ml of medium 199 with or without Listeria was injected subcutaneously in a shaved abdominal area with a 20-gauge, 1-inch (ca. 2.5-cm) needle. Intratumoral injections of Listeria were performed by using a volume of 0.2 ml of saline and a 26-gauge needle. The needle was inserted into the tumor mass with the bevel up, and the entire tumor was irrigated by rotating the needle in a 1800 arc. The aniimals were monitored for tumor growth at regular intervals over a 3-month period. The size of the tumor is expressed as the mean tumor diameter computed after measuring two perpendicular tumor diameters. Preparation of tumor antigen. A 3 M KCI extract of the tumor was prepared by the method of Meltzer et al. (19) by using 2 ml of 3 M KCI in 0.005 M potassium phosphate buffer (pH 7.2) per g of tumor. The antigen solution thus obtained was assayed for protein concentration by the biuret test, distributed into vials, and lyophilized. Measurements and evaluation of skin reactions. Skin reactions were measured by using the following three criteria: (i) degree of erythema, (ii) mean diameter of reaction, and (iii) changes in the double thickness of skin at the site of reaction. Double skin thickness was evaluated with a Schnelltaster as described previously (5). These three criteria were arbitrarily evaluated as shown in Table 1.
55
percent mortality and also gave the most uniform pattern of growth. The tumor grew as a localized subcutaneous mass which ulcerated through the skin. Penetration of the peritoneal cavity was a very rare occurrence. Lung metastases were present at autopsy in about 75% of the animals. Effect of Listeria on the growth of MCA1 cells. (i) Listeria inoculated in mixture with tumor cells. Listeria cells were injected with 2.5 x 106 MCA-1 cells into strain 13 guinea pigs in ratios of Listeria to tumor cells of 1:100, 1:200, and 1:400. Animals injected with tumor cells alone served as controls. Figures 1 through TABLE 2. Incidence of mortality in guinea pigs injected with different doses of MCA-1 cells Tumor cell No. dead/no. Survival timea dose injected (days) 1 x 107 6/10 38, 45, 45, 48, 55, 86 5 x 106 7/10 24, 41, 54, 61, 79, 83, 106 2.5 x 106 8/10 61, 61, 61, 65, 66, 75, 79, 79 1 x 106 5/10 49,61,75,75,75 5 x 10 3/9 59, 59, 89 a Animals were kept under observation for 120 days. The remaining animals in each group were alive and tumor-free at the end of the observation period. 45.0
RESULTS Growth characteristics of different subcutaneous doses of MCA-1 tumor cells. To determine the lowest dose of MCA-1 cells which would lead to progressive tumor growth in strain 13 guinea pigs, groups of 10 sex-matched animals were injected subcutaneously with 1 x 107, 5 x 106, 2.5 x 106, 1 x 106, or 5 x 105 MCA-1 tumor cells. Table 2 shows the survival times of the progressor. Up to a certain point the mortality rate was dose dependent. With doses above 2.5 x 106 cells, however, there was no marked increase in mortality. The dose of 2.5 x 106 cells was therefore chosen for future experiments because it was the lowest dose which resulted in the highest
40.0 35.0-
2-30.0* 25.0-
° 20.0
{l
Z 15.0 La
5.0
TABLE 1. Evaluation of skin reactions' Score
Erythema
Diameter (mm)
Double skin thickness (mm x 10)
