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Ehrlich ascites tumor growth in gnotobiotic mice
Ehrlich Ascites Tumor Growth in Gnotobiotic JAMES
B. HENEGHAN,
PH.D.,
From the Department of Surgery, Louisiana State University Medical Center, New Orleans, Louisiana. This work mas supported by American Cancer Society Grant T-327 and U.S.P.H.S. Grant FR-00272 from the Dinision of Research Facilities and Resources of the National Institutes of Health.
New Orleans, Louisiana
Once the ascites tumor was established and maintained in germ-free mice, the effects of microorganisms on tumor growth were studied. MATERIAL
Establishment free Animals.
AND METHODS
of Ehrlich
Ascites
Tumor
in Germ-
All of the mice utilized in these experiments were L.S.U. strain CFW Swiss mice of either sex which weighed 25 to 35 gm. at approximately seventy-five days of age. All animals were maintained in clear plastic mouse cages on corn cob bedding (San-i-eel@)and were fed ad lib&urnwater and diet (Purina@ 5010 C) which was sterilized for twenty-five minutes at 252’~. in the standardsterilizing cylinders. The gnotobiotic animals were maintained in flexible film isolators whereas the conventional animals were maintained in the open laboratory. The initial Ehrlich ascites tumor cells were obtained in 1961 and maintained in the conven-
our interest in the relationship of bacteria to tumor growth was stimulated when studies with the Brown-Pearce tumor in rabbits showed that the use of antibiotics to control the microbial flora of the colon prior to surgery resulted in an increased incidence of tumor growth at the site of gastrointestinal anastomosis [I]. From this observation it may be suspected that certain elements are present within the so-called normal intestinal flora which inhibit tumor growth. Actually other investigators have reported that certain bacteria have a beneficial effect on established tumors in oiuo. Koshimura et al. [Z] found a fourfold increase in survival when mice were inoculated intraperitoneally with Ehrlich ascites carcinoma cells and living hemolytic streptococci. M&e and M&e [3 ] have reported the regression of solid Ehrlich carcinoma in mice after intravenous injection of nonpathogenic clostridia spores. However, these investigators were quick to point out that due to the toxic effects of the accelerated tumor necrosis there was no increase in survival. The purpose of this investigation was to establish the growth of a sterile preparation of a well known tumor cell line in germ-free animals. The Ehrlich ascites tumor was chosen because of the ease of transplantation and the probability that the tumor would be sterile in the peritoneal cavity of conventional animals.
I
Mice*
NITIALLY
tional mouse colony. After rigorous surgical preparation of the abdomen, tumor cells were aseptically aspirated from the peritoneal cavity of a conventional mouse into a sterile heparinized syringe. The cells were then sealed into a sterilized glass ampule which was sprayed into the isolator with peracetic acid. Initially only a few mice were placed into the isolator and inoculated. Later after cultures of the tumor cells, the isolator, and the animal cxcreta had confirmed that the cells were sterile and that the transfer of cells had been accomplished without contamination, other germ-free mice were inoculated. During the two year period of this investigation approximately 250 mice or one third of all the mice in the tumor study were utilized in maintaining the
tumor cell line within the germ-freeisolation system. Experimental Procedures. One week after inoculation donor animals were sacrificed and the heparinized tumor cells were pooled and divided into equal aliquots for each experimental category, passed into the appropriate isolators, and brought outside for
* Presented at the Eighth Annual Meeting of the Society for Surgery of the Alimentary Tract, Atlantic City, New Jersey, June 17 and 18, 1967. 108
American
Journal of Surgery
Tumor Growth in Gnotobiotic Mice TABLE EHRLICH
ASCITES
Ko. of Survivors Among No. Inoculated
TUMOR
GROWTH
IN
I
GNOTOBIOTIC
Status
MICE
DAY
GROWTH)
Total Packed Cell Volume (ml.)
Fluid Weight (gm.)
Ascitocrit of Cells (ye)
2.93
___~
Conventional Germ-free Staph. aureus
9.8
8.8 8.6
30 30 22
21/25 19/25 16/25
Conventional Germ-free B. cereus
9.4 8.2 10.3
23 26 24
2.14 2.14 2.49
lO/lO 9/10 9/10 lO/lO
Conventional Germ-free “Old” Staph. aureus “New” Staph. aureus
8.5 8.3 8.4 8.8
16 16 12 17
1.92 1.87 1.47 1.92
TABLE EHRLJCH
Status
Conventional Germ-free “Old” Staph. aureus “New” Staph. aureus
January
1968
2.67 1.86
after colonization and have been indicated in this report by the prefix “new.” In the studies designed to suppress the reticuloendothelial system, mice were weighed and given three daiiy dosages of 200 mg./kg. of azathioprine by means of a stomach tube. This is a total dosage of 600 mg./kg. for each mouse which is the LDw dose for conventional mice but is 150 mg./kg. below the LDa, dose for germ-free mice [5]. Five days after the last dosage of azathioprine, the mice were challenged with tumor and ten days later tumor growth was determined. White blood cell counts were obtained as an indication of the status of the reticuloendothelial system before and after immunosuppressive therapy. RESULTS
The data in Table I indicate that the growth of the Ehrlich ascites tumor was similar in both germ-free and conventional animals as indicated by the total packed cell volume. All 150 animals in the first experiment were inoculated with aliquots of the one pooled tumor cell suspension which resulted in a total packed cell volume of 2.93 ml. in conventional animals. All II
CARCINOMA
STUDIES
Ten Day Growth after Scapular Inoculation
115.
(TEN
37/50 36/50 43/50
inoculation in conventional animals. Germ-free and conventional control groups were included in each experiment involving monocontaminated animals. An aliquot of the cells removed from the isolator for inoculation into conventional animals was used for a cell count to determine the amount of inoculum necessary for a dose of 5 million cells per mouse. Animals in each experimental category were sacrificed and weighed before and after the removal of the ascites fluid to determine the weight or amount of fluid present. Aliquots of the tumor cell suspension were taken for cell counts and for determination of the ascitocrit, the percentage of tumor cells in the ascites fluid. The product of the amount of fluid and the ascitocrit is called the total packed cell volume and is a reliable parameter of tumor growth [d]. In the studies with monocolonization, twenty-four hour cultures of either Bacillus cereus ATCC No. 9634 or Staphylococcus aureus ATCC No. 6538P were grown in screw cap tubes which were sprayed into the isolators with peracetic acid. Each mouse was colonized by swabbing its oral cavity with microorganisms. In addition pellets of food and the drinking water were also inoculated. Animals were then challenged with tumor six weeks later. Some animals were challenged with tumor immediately
Vol.
109
Per Cent of Survival after Intraperitoneal Inoculation
No. with Tumor Among No. Inoculated
Average Weight of Tumor (gm.)
No. Inoculated
5/13 5/9 o/10 4/11
0.70 0.40 0.00 0.44
15 15 15 15
Weeks after Inoculation 2
4
6
87 100 100 100
20 27 53 33
0 0 20 0
Heneghan
110
TABLE III EHRLICHA~~ITE~TUMORGRO~THIN MICE TENDAYSAFTERTUMORCELLIN~CULATION AND FIFTEENDAYSAFTERAZATHIOPRINE THERAPY
No. of Animals
Status
Conventional Untreated Treated Germ-free Untreated Treated Staph. aureus Untreated Treated
10 9 11 10 10 14 10 9 9
White Blood Cell Count (cells per mma.) Control
At Sacrifice
Tumor Growth at Sacrifice as Measured by Total Packed Cell Volume (ml.)
26,000 24,000
2.08 1.44
16,100 4,100
1.98 1.38
22 ) 000 6,000
1.51 1.33
8,700
6,050
12,000
forty animals in the third experiment were also inoculated with aliquots of another pooled collection of tumor cells which resulted in a total packed cell volume of 1.92 ml. in conventional animals. It can be readily seen from the variability of these data that each experiment with a single cell pool must have both conventional and germ-free control groups and the results compared with those of other experiments in values relative to the controls. Colonization of germ-free mice with Staph. aureus (Table I) inhibited tumor growth since there was a 33 per cent reduction in the total packed cell volume. Also, a similar reduction occurred in the ascitocrit or percentage of tumor cells in the peritoneal fluid. This indicated an actual reduction in the number of tumor cells present as confirmed by actual cell counts. This inhibition was significant at the 5 per cent level in the T test. Colonization with B. cereus, on the other hand, produced a slight increase in tumor growth which was not statistically significant. In the third experimental series (Table I) the Staph. aureus category was divided into two groups; one group which was colonized six weeks prior to tumor challenge was called the “old” Staph. aureus group and the other which was colonized immediately prior to tumor challenge was called the “new” Staph. aureus group. Colonization with simultaneous tumor challenge produced no inhibition of tumor growth, yet an inhibition was observed in the mice which had been colonized for six weeks. The ineffectiveness of simultaneous colonization and tumor challenge was again observed in the per cent of survival after intraperitoneal inoculation (Table II) and in the per cent of tumor
takes after subcutaneous inoculation in the scapular region. Another group of experiments was performed to determine whether the inhibition of tumor growth observed after a six week colonization with Staph. aureus was due to stimulation of the hypoactive reticuloendothelial system of germ-free animals. The results of the experiments with azathioprine therapy are shown in Table III. Control white blood cell counts in the Staph. aureus monocolonized animals were significantly higher than those in the germ-free mice. However, after treatment with azathioprine the white blood cell counts of mice in both groups were in the same range. Also, azathioprine therapy eliminated the inhibition of tumor growth observed in the untreated Staph. aureus colonized mice. Azathioprine therapy not only suppressed the bone marrow (lower white blood cell counts) but also inhibited all cell division (lower total packed cell volume in each treated group). Since all of the determinations of tumor cell growth are based on the weight of the ascitic fluid, it was thought that these data should be related to the corrected body weight of the mice. When this factor was employed, to eliminate possible variables due to the enlarged cecum of germ-free rodents, the same relationships existed, that is, a 30 to 33 per cent reduction in tumor growth due to Staph. aureus monocolonization. COMMENTS
The successful aseptic transplantation of Ehrlich ascites tumor into germ-free mice, which was accomplished during the initial phase American
Journal of Surgery
Tumor Growth in Gnotobiotic Mice of this project, represented one of the first transplantations of viable cancer cells into germ-free animals. Miyakawa et al. [6] studied the transplantation of three different types of tumor cells, that is, Uoshida sarcoma, human cancer cells, and human leukemic cells, into germ-free guinea pigs. The growth of the Ehrlich ascites tumor and the Ehrlich subcutaneous scapular carcinoma in germ-free and conventional mice was similar in both bacteriologic categories in every experimental series. Each time the germ-free mice were colonized with a pure culture of Staph. aureus for at least six weeks, a 30 per cent reduction in tumor growth was observed. However, simultaneous challenge with Staph. aureus and tumor cells did not produce any inhibition in tumor growth. These observations lead to the conclusion that the inhibitory response is not a direct effect of the bacteria on the tumor cells but rather an indication of some change which occurs in the host animal during a six week colonization period. Finally it was observed that the Staph. aureus colonized mice had a high white blood cell count prior to azathioprine treatment when tumor growth was inhibited but a low white blood cell count after immunosuppressive therapy which eliminated the tumor growth inhibiting effect of colonization. This suggests that the hypoactive reticuloendothelial system of the germ-free animal was stimulated by a six week association with this organism and, thus, when the animals were challenged with tumor, there were more reticuloendothelial elements present to attack these foreign bodies. CONCLUSIONS
1. A well defined tumor cell line, the Ehrlich ascites tumor, was established in germ-free mice. 2. Growth of this tumor was similar in germfree and conventional mice. 3. Prior monocolonization with Staphylococcus aureus for six weeks reduced tumor growth approximately 30 per cent. 4. White blood cell counts and studies with an immunosuppressive agent suggest that the
Vol. 115. January
1968
111
inhibition of tumor growth was due to stimulation of the hypoactive reticuloendothelial system of the germ-free mouse. REFERENCES
1. COHN, I., JR. and ATIK, M. The influence of antibiotics on the spread of tumor of the colon. An experimental study. Ann. Surg., 151: 917, 1960. 2. KOSHIMURA, S., MUROSAWA, K., NAKAGAWA, E., UEDA, M., BANDO, Y., and HIRATA, R. Experimental anticancer studies. III. On the influence of living hemolytic streptococci upon the invasion power of Ehrlich ascites carcinoma in mice. Jup. J. Exper. Med., 25: 93, 1955. 3. M&E, J. R. and MGsE, G. Oncolysis by clostridia. I. Activity of Clostridium butyricum (M-55) and other nonpathogenic clostridia against the Ehrlich carcinoma. Cancer Res., 24: 212, 1964. 4. SASSENRATH, E. N., Total packed cell volume as a measure of growth retardation in mouse ascites tumors. Ann. New York Acad. Sci., 76: 601, 1958. 5. NANCE, C. F., SILAS, D. J., and HENEGHAN, J. B. Changes in azathioprine toxicity in gnotobiotic mice. S. Forum, 1967. Submitted for publication. 6. MIYAKAWA, M., KISHIMOTO, H., ITAYA, J., UEI, Y., UNO, Y., and MIYAZU, M. Experimental studies on the transplantation of Yoshida sarcoma, human cancer, and human leukemic cells into germfree guinea pigs. Jap. J. Cancer Res., 48: 530, 1957. DISCUSSION JAMES B. HENEGHAN (New Orleans, La.): I would like to make one comment about the future of these tumor growth experiments in germ-free mice. Recently, sometime after we began these experiments and became interested in the effects of bacteria on tumor growth, some investigators at Notre Dame were able to demonstrate the presence of viruses in at least six different strains of germfree mice. They unmasked these viruses by using sublethal doses of roentgen radiation, actually 50 rads given one week apart for four weeks, and after six weeks lymphatic leukemia developed in all of the strains. By electron microscopy they could demonstrate viruses in the thymus glands and were also able to transmit the disease with cell-free filtrates of the thymus gland without irradiation. No one ever claimed these animals were virusfree and it appears as though the mice are not. Using the same technics in germ-free rats, it has not been possible to date to demonstrate any viruses, although various leukemias and other neoplastic diseases have been stimulated primarily by chemical carcinogens in these animals.
B. HENEGHAN,
PH.D.,
From the Department of Surgery, Louisiana State University Medical Center, New Orleans, Louisiana. This work mas supported by American Cancer Society Grant T-327 and U.S.P.H.S. Grant FR-00272 from the Dinision of Research Facilities and Resources of the National Institutes of Health.
New Orleans, Louisiana
Once the ascites tumor was established and maintained in germ-free mice, the effects of microorganisms on tumor growth were studied. MATERIAL
Establishment free Animals.
AND METHODS
of Ehrlich
Ascites
Tumor
in Germ-
All of the mice utilized in these experiments were L.S.U. strain CFW Swiss mice of either sex which weighed 25 to 35 gm. at approximately seventy-five days of age. All animals were maintained in clear plastic mouse cages on corn cob bedding (San-i-eel@)and were fed ad lib&urnwater and diet (Purina@ 5010 C) which was sterilized for twenty-five minutes at 252’~. in the standardsterilizing cylinders. The gnotobiotic animals were maintained in flexible film isolators whereas the conventional animals were maintained in the open laboratory. The initial Ehrlich ascites tumor cells were obtained in 1961 and maintained in the conven-
our interest in the relationship of bacteria to tumor growth was stimulated when studies with the Brown-Pearce tumor in rabbits showed that the use of antibiotics to control the microbial flora of the colon prior to surgery resulted in an increased incidence of tumor growth at the site of gastrointestinal anastomosis [I]. From this observation it may be suspected that certain elements are present within the so-called normal intestinal flora which inhibit tumor growth. Actually other investigators have reported that certain bacteria have a beneficial effect on established tumors in oiuo. Koshimura et al. [Z] found a fourfold increase in survival when mice were inoculated intraperitoneally with Ehrlich ascites carcinoma cells and living hemolytic streptococci. M&e and M&e [3 ] have reported the regression of solid Ehrlich carcinoma in mice after intravenous injection of nonpathogenic clostridia spores. However, these investigators were quick to point out that due to the toxic effects of the accelerated tumor necrosis there was no increase in survival. The purpose of this investigation was to establish the growth of a sterile preparation of a well known tumor cell line in germ-free animals. The Ehrlich ascites tumor was chosen because of the ease of transplantation and the probability that the tumor would be sterile in the peritoneal cavity of conventional animals.
I
Mice*
NITIALLY
tional mouse colony. After rigorous surgical preparation of the abdomen, tumor cells were aseptically aspirated from the peritoneal cavity of a conventional mouse into a sterile heparinized syringe. The cells were then sealed into a sterilized glass ampule which was sprayed into the isolator with peracetic acid. Initially only a few mice were placed into the isolator and inoculated. Later after cultures of the tumor cells, the isolator, and the animal cxcreta had confirmed that the cells were sterile and that the transfer of cells had been accomplished without contamination, other germ-free mice were inoculated. During the two year period of this investigation approximately 250 mice or one third of all the mice in the tumor study were utilized in maintaining the
tumor cell line within the germ-freeisolation system. Experimental Procedures. One week after inoculation donor animals were sacrificed and the heparinized tumor cells were pooled and divided into equal aliquots for each experimental category, passed into the appropriate isolators, and brought outside for
* Presented at the Eighth Annual Meeting of the Society for Surgery of the Alimentary Tract, Atlantic City, New Jersey, June 17 and 18, 1967. 108
American
Journal of Surgery
Tumor Growth in Gnotobiotic Mice TABLE EHRLICH
ASCITES
Ko. of Survivors Among No. Inoculated
TUMOR
GROWTH
IN
I
GNOTOBIOTIC
Status
MICE
DAY
GROWTH)
Total Packed Cell Volume (ml.)
Fluid Weight (gm.)
Ascitocrit of Cells (ye)
2.93
___~
Conventional Germ-free Staph. aureus
9.8
8.8 8.6
30 30 22
21/25 19/25 16/25
Conventional Germ-free B. cereus
9.4 8.2 10.3
23 26 24
2.14 2.14 2.49
lO/lO 9/10 9/10 lO/lO
Conventional Germ-free “Old” Staph. aureus “New” Staph. aureus
8.5 8.3 8.4 8.8
16 16 12 17
1.92 1.87 1.47 1.92
TABLE EHRLJCH
Status
Conventional Germ-free “Old” Staph. aureus “New” Staph. aureus
January
1968
2.67 1.86
after colonization and have been indicated in this report by the prefix “new.” In the studies designed to suppress the reticuloendothelial system, mice were weighed and given three daiiy dosages of 200 mg./kg. of azathioprine by means of a stomach tube. This is a total dosage of 600 mg./kg. for each mouse which is the LDw dose for conventional mice but is 150 mg./kg. below the LDa, dose for germ-free mice [5]. Five days after the last dosage of azathioprine, the mice were challenged with tumor and ten days later tumor growth was determined. White blood cell counts were obtained as an indication of the status of the reticuloendothelial system before and after immunosuppressive therapy. RESULTS
The data in Table I indicate that the growth of the Ehrlich ascites tumor was similar in both germ-free and conventional animals as indicated by the total packed cell volume. All 150 animals in the first experiment were inoculated with aliquots of the one pooled tumor cell suspension which resulted in a total packed cell volume of 2.93 ml. in conventional animals. All II
CARCINOMA
STUDIES
Ten Day Growth after Scapular Inoculation
115.
(TEN
37/50 36/50 43/50
inoculation in conventional animals. Germ-free and conventional control groups were included in each experiment involving monocontaminated animals. An aliquot of the cells removed from the isolator for inoculation into conventional animals was used for a cell count to determine the amount of inoculum necessary for a dose of 5 million cells per mouse. Animals in each experimental category were sacrificed and weighed before and after the removal of the ascites fluid to determine the weight or amount of fluid present. Aliquots of the tumor cell suspension were taken for cell counts and for determination of the ascitocrit, the percentage of tumor cells in the ascites fluid. The product of the amount of fluid and the ascitocrit is called the total packed cell volume and is a reliable parameter of tumor growth [d]. In the studies with monocolonization, twenty-four hour cultures of either Bacillus cereus ATCC No. 9634 or Staphylococcus aureus ATCC No. 6538P were grown in screw cap tubes which were sprayed into the isolators with peracetic acid. Each mouse was colonized by swabbing its oral cavity with microorganisms. In addition pellets of food and the drinking water were also inoculated. Animals were then challenged with tumor six weeks later. Some animals were challenged with tumor immediately
Vol.
109
Per Cent of Survival after Intraperitoneal Inoculation
No. with Tumor Among No. Inoculated
Average Weight of Tumor (gm.)
No. Inoculated
5/13 5/9 o/10 4/11
0.70 0.40 0.00 0.44
15 15 15 15
Weeks after Inoculation 2
4
6
87 100 100 100
20 27 53 33
0 0 20 0
Heneghan
110
TABLE III EHRLICHA~~ITE~TUMORGRO~THIN MICE TENDAYSAFTERTUMORCELLIN~CULATION AND FIFTEENDAYSAFTERAZATHIOPRINE THERAPY
No. of Animals
Status
Conventional Untreated Treated Germ-free Untreated Treated Staph. aureus Untreated Treated
10 9 11 10 10 14 10 9 9
White Blood Cell Count (cells per mma.) Control
At Sacrifice
Tumor Growth at Sacrifice as Measured by Total Packed Cell Volume (ml.)
26,000 24,000
2.08 1.44
16,100 4,100
1.98 1.38
22 ) 000 6,000
1.51 1.33
8,700
6,050
12,000
forty animals in the third experiment were also inoculated with aliquots of another pooled collection of tumor cells which resulted in a total packed cell volume of 1.92 ml. in conventional animals. It can be readily seen from the variability of these data that each experiment with a single cell pool must have both conventional and germ-free control groups and the results compared with those of other experiments in values relative to the controls. Colonization of germ-free mice with Staph. aureus (Table I) inhibited tumor growth since there was a 33 per cent reduction in the total packed cell volume. Also, a similar reduction occurred in the ascitocrit or percentage of tumor cells in the peritoneal fluid. This indicated an actual reduction in the number of tumor cells present as confirmed by actual cell counts. This inhibition was significant at the 5 per cent level in the T test. Colonization with B. cereus, on the other hand, produced a slight increase in tumor growth which was not statistically significant. In the third experimental series (Table I) the Staph. aureus category was divided into two groups; one group which was colonized six weeks prior to tumor challenge was called the “old” Staph. aureus group and the other which was colonized immediately prior to tumor challenge was called the “new” Staph. aureus group. Colonization with simultaneous tumor challenge produced no inhibition of tumor growth, yet an inhibition was observed in the mice which had been colonized for six weeks. The ineffectiveness of simultaneous colonization and tumor challenge was again observed in the per cent of survival after intraperitoneal inoculation (Table II) and in the per cent of tumor
takes after subcutaneous inoculation in the scapular region. Another group of experiments was performed to determine whether the inhibition of tumor growth observed after a six week colonization with Staph. aureus was due to stimulation of the hypoactive reticuloendothelial system of germ-free animals. The results of the experiments with azathioprine therapy are shown in Table III. Control white blood cell counts in the Staph. aureus monocolonized animals were significantly higher than those in the germ-free mice. However, after treatment with azathioprine the white blood cell counts of mice in both groups were in the same range. Also, azathioprine therapy eliminated the inhibition of tumor growth observed in the untreated Staph. aureus colonized mice. Azathioprine therapy not only suppressed the bone marrow (lower white blood cell counts) but also inhibited all cell division (lower total packed cell volume in each treated group). Since all of the determinations of tumor cell growth are based on the weight of the ascitic fluid, it was thought that these data should be related to the corrected body weight of the mice. When this factor was employed, to eliminate possible variables due to the enlarged cecum of germ-free rodents, the same relationships existed, that is, a 30 to 33 per cent reduction in tumor growth due to Staph. aureus monocolonization. COMMENTS
The successful aseptic transplantation of Ehrlich ascites tumor into germ-free mice, which was accomplished during the initial phase American
Journal of Surgery
Tumor Growth in Gnotobiotic Mice of this project, represented one of the first transplantations of viable cancer cells into germ-free animals. Miyakawa et al. [6] studied the transplantation of three different types of tumor cells, that is, Uoshida sarcoma, human cancer cells, and human leukemic cells, into germ-free guinea pigs. The growth of the Ehrlich ascites tumor and the Ehrlich subcutaneous scapular carcinoma in germ-free and conventional mice was similar in both bacteriologic categories in every experimental series. Each time the germ-free mice were colonized with a pure culture of Staph. aureus for at least six weeks, a 30 per cent reduction in tumor growth was observed. However, simultaneous challenge with Staph. aureus and tumor cells did not produce any inhibition in tumor growth. These observations lead to the conclusion that the inhibitory response is not a direct effect of the bacteria on the tumor cells but rather an indication of some change which occurs in the host animal during a six week colonization period. Finally it was observed that the Staph. aureus colonized mice had a high white blood cell count prior to azathioprine treatment when tumor growth was inhibited but a low white blood cell count after immunosuppressive therapy which eliminated the tumor growth inhibiting effect of colonization. This suggests that the hypoactive reticuloendothelial system of the germ-free animal was stimulated by a six week association with this organism and, thus, when the animals were challenged with tumor, there were more reticuloendothelial elements present to attack these foreign bodies. CONCLUSIONS
1. A well defined tumor cell line, the Ehrlich ascites tumor, was established in germ-free mice. 2. Growth of this tumor was similar in germfree and conventional mice. 3. Prior monocolonization with Staphylococcus aureus for six weeks reduced tumor growth approximately 30 per cent. 4. White blood cell counts and studies with an immunosuppressive agent suggest that the
Vol. 115. January
1968
111
inhibition of tumor growth was due to stimulation of the hypoactive reticuloendothelial system of the germ-free mouse. REFERENCES
1. COHN, I., JR. and ATIK, M. The influence of antibiotics on the spread of tumor of the colon. An experimental study. Ann. Surg., 151: 917, 1960. 2. KOSHIMURA, S., MUROSAWA, K., NAKAGAWA, E., UEDA, M., BANDO, Y., and HIRATA, R. Experimental anticancer studies. III. On the influence of living hemolytic streptococci upon the invasion power of Ehrlich ascites carcinoma in mice. Jup. J. Exper. Med., 25: 93, 1955. 3. M&E, J. R. and MGsE, G. Oncolysis by clostridia. I. Activity of Clostridium butyricum (M-55) and other nonpathogenic clostridia against the Ehrlich carcinoma. Cancer Res., 24: 212, 1964. 4. SASSENRATH, E. N., Total packed cell volume as a measure of growth retardation in mouse ascites tumors. Ann. New York Acad. Sci., 76: 601, 1958. 5. NANCE, C. F., SILAS, D. J., and HENEGHAN, J. B. Changes in azathioprine toxicity in gnotobiotic mice. S. Forum, 1967. Submitted for publication. 6. MIYAKAWA, M., KISHIMOTO, H., ITAYA, J., UEI, Y., UNO, Y., and MIYAZU, M. Experimental studies on the transplantation of Yoshida sarcoma, human cancer, and human leukemic cells into germfree guinea pigs. Jap. J. Cancer Res., 48: 530, 1957. DISCUSSION JAMES B. HENEGHAN (New Orleans, La.): I would like to make one comment about the future of these tumor growth experiments in germ-free mice. Recently, sometime after we began these experiments and became interested in the effects of bacteria on tumor growth, some investigators at Notre Dame were able to demonstrate the presence of viruses in at least six different strains of germfree mice. They unmasked these viruses by using sublethal doses of roentgen radiation, actually 50 rads given one week apart for four weeks, and after six weeks lymphatic leukemia developed in all of the strains. By electron microscopy they could demonstrate viruses in the thymus glands and were also able to transmit the disease with cell-free filtrates of the thymus gland without irradiation. No one ever claimed these animals were virusfree and it appears as though the mice are not. Using the same technics in germ-free rats, it has not been possible to date to demonstrate any viruses, although various leukemias and other neoplastic diseases have been stimulated primarily by chemical carcinogens in these animals.