Brief Communication: Delayed Lethal Response to Candida alblcans Infection in Mice Bearing the Lewis Lung Carcinoma 1, 2 E. H. Robinette, Jr., and D. N. Mardon
3, 4
SUMMARY-Lethality of Candida albicans was monitored in (C57BL x DBA/2)F1 mice bearing the transplanted Lewis lung carcinoma and in sham-operated controls inoculated with Hanks' balanced salt solution_ The lethal response to C. albicans infection was significantly delayed in animals inoculated with the microorganism 6-16 days after transplantation of the Lewis lung carcinoma. Maximal increases in survival times were observed when C. albicans was inoculated 8-12 days following tumor transplantation. Therefore, a delay in the lethal response to C. albicans in this untreated model murine tumor system could be elicited through implantation of the Lewis lung carci· noma; preliminary studies with some other model murine tumors and with cell-free filtrates indicated that this phenom· enon is not restricted to the Lewis lung carcinoma.-J Natl Cancer Inst 55: 731-733, 1975.
The microorganism used throughout this investigation was C. albicans (ATCC #10261), maintained and cultured as previously described (12). Sterile 0.85% saline suspensions of washed yeastlike C. albicans preparations were adjusted to predetermined turbidity (#66 red filter) with a Klett-Summerson colorimeter. These cultures averaged 1 X 101 cells/m!. In all experiments 0.5 ml of the suspension (for control animals, 0.85% saline) was inoculated iv into a lateral tail vein of each mouse. Following inoculation with C. albicans the mice were observed daily and mortality rates recorded. Animals were routinely examined at death for gross pathology. Mouse mortality rates were evaluated statistically by the Wilcoxon method (13). RESULTS AND DISCUSSION
Opportunistic candidiasis occurs in cancer patients and in naturally or artificially immunosuppressed hosts (1-5). However, it is uncertain whether candidiasis results primarily from an intrinsic defect in host resistance caused by the cancer or from immunosuppression caused by anticancer therapy. These clinical problems have stimulated attempts to define the factors influencing host resistance to this microorganism (6-8). However, there have been no reports in the literature concerning host resistance to Candida albicans in a model tumor system. In the present study, we examined the response of mice bearing the Lewis lung carcinoma (LLC) to challenge with a lethal injection of C. albicans in the absence of antitumor or antimicrobial therapy. MATERIALS AND METHODS
(C57BL X DBA/2)Fl (BDFl) mice were obtained from The Jackson Laboratory, Bar Harbor, Maine. At the start of each experiment, the animals were 6 weeks old and weighed 17-22 g. The transplanted LLC originated from a spontaneous (anaplastic) carcinoma discovered in the lung of a C57BL mouse by Margaret R. Lewis in 1951 (9). This tumor, which metastasizes rapidly to the lung (10), was maintained and transplanted sc every 16-18 days in the syngeneic C57BL/6J mouse, as described by Mayo (11). All subsequent experiments were done with BDFI mice. A I-mm2 fragment of the LLC was implanted sc into the left hindquarter of each BDFI mouse receiving a tumor implant. Sham operations were done on anesthesized BDFI mice (controls), and these animals were then inoculated sc with 0.1 ml Hanks' balanced salt solution (HBS). In some experiments, shamoperated BDFI mice were inoculated sc with 0.1 ml MiIIipore filtrate (0.45 nm porosity) from minced LLC cells harvested 16-18 days after transplantation into C57BL/6J mice. This LLC filtrate (LLF) was prepared by aseptic suspension of 60 pieces of tumor (::::::1.0 mm 2 each) in 10 ml sterile HBS. The preparation was filtered (MiIlipore), and 0.1 ml was implanted sc into each mouse at the site of the incision. No mice inoculated with the filtrate developed tumors within 60 days.
A significant delay in the lethal response to C. albicans in BDFI mice bearing transplanted LLC occurred when mice were challenged with this microorganism 8 days after tumor implantation (text-fig. I). Conversely, when mice were infected with C. albicans 0, 2, or 4 days following implantation of LLC fragments, no significant delay in mortality was observed (table 1). Maximal increased survival times occurred when C. albicans was injected into mice 8-12 days after tumor implantation. Control mice bearing only the carcinoma began to die by day 18; therefore, reliable data could not be obtained beyond 16 days. Injection of C. albicans into BDFI mice 8 days after implantation of LLC fragments, other model murine tumors, or cell-free filtrates from the tumors (table 2) indicated that this phenomenon, though not universal, is not unique to the LLC system. Furthermore, these preliminary data suggest that a delayed lethal response similar to that observed with tumor fragments can be obtained with Millipore filtrates from tumor preparations. This effect was not seen when organ filtrates from normal BDFl mice were used in place of tumor filtrates. Thus experiments suggest that the activity against C. albicans does not require transplantation of tumor cells per se but depends on a component from the malignant cells. The availability of Millipore filtrates as suitable mediators of a delayed lethal response to C. albicans should allow us to extend our studies into the nature of the response to this microorganism elicited in the tumor-bearing host. Since the filtrates used in these experiments were prepared from tumor cells harvested only once after implantation, it is presently unknown whether material with greater activity can be prepared from tumor cell filtrates obtained at different intervals during carcinogenesis. Received April I, 1975; accepted May 30, 1975. Supported in part by Public Health Service grant CAI6627 from the National Cancer Institute and by grant ACS IN-105 from the American Cancer Society. 3 Department of Microbiology, Box 847, Medical College of Virginia, Virginia Commonwealth University, Richmond, Va. 23298. 4 We thank Ms. Trisha Reed for her technical assistance, and Drs. A. E. Munson, A. M. Kaplan, and P. S. Morahan for their helpful discussions. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.3, SEPTEMBER 1975
731
732
ROBINETTE AND MARDON
pared with LLC-bearing mice and those receiving LLF (text-fig. 2). 100
TABLE
2.-Mean survival times for mice inoculated with C. albicans
8 days after implantation with murine tumors and Millipore
filtrates from tumor preparations
80
Implant a
Survival (days) b
Tumor-bearing/ control c
60
Lewis lung carcinoma ___________ Millipore filtrate _______________ HBS controL _________________
7.3±OA 5.9±0.5 4.1±O.7
178 144 100
40
B-16 melanotic melanoma _______ Millipore filtrate _______________ HBS controL _________________
7.6±0.S 5.6±O.5 4.0±O.6
190 141 100
3-Methylcholanthrene-induced fibrosarcoma-l d Millipore filtrate _______________ HBS controL __________________
7A±0.6
20
186
6.S±0.6 4.0±0.6
170 100
6.6±0.7
124
5.7±0.4 5.3±0.4
106 100
III III:
0
>
>
III:
:::l
III ~
z
""U ""a..
III:
12
8
14
3-Methylcholanthrene-induced fibrosarcoma-2 d Millipore filtrate _______________ HBS controL _________________
16
DAYS POST IWOCULATION I.-Mortality from C. albicans in groups of mice bearing LLC or in groups of sham-operated control mice receiving HBS. Each animal was inoculated 8 days (vertical dotted line) after LLC or HBS implantation, with 0.5 ml standard cell suspension of yeastlike C. albicans. Curves represent data from 3 experiments, each consisting of experimental and control groups of 10 mice each. Variation among the 3'experiments: P:S;0.05.
TEXT-FIGURE
TABLE
I.-Mean survival times for mice inoculated with C. albicans at selected intervals following LLC implantation
Day post implantation a
0 2 4 6 8 10 12 14 16
Survival (days)
b
LLC
HBS
4.5±OA 5.3±O.8 4.8±0.8 6.3±O.8 7.3±0.4 8.7±1.0 7.8±0.6 8.2±1.1 6.8 ± 1.1
4.2±O.6 4.7±O.7 4.5±0.7 4.2±0.6 4.1±0.7 5.0±1.0 4A±0.4 5.0±1.0 4A±1.1
• All experimental and control BDFI mice received sc in the left hindquarter appropriate tumor fragments, filtrate from minced tumor cells prepared as described in "Materials and Methods." or HB8. Each value represents data from a group of 16 BDF, mice. All tumors were obtained from C57BL mice. Eight days after implantation, each mouse was challenged iv with 5 X 106 yeastIike cells of C.1l1bicans. • Mean survival after infection with C. albicllns ±SE. , Percent ratio of mean survival days for tumor-bearing/control mice. Significant increase in survival occured at values? 140. d 3-Methylcholanthrene-induced fibrosarcoma-l was maintained via multiple serial transplants in C57BL mice. 3-Methylcholanthrene-induced fibrosarcoma-2 was maintained frozen in liquid nitrogen and used after the first transplant into C57BL mice.
Tumor-bearing/ control c
15
107 113
107 150 180 174 177 164 154
• Following sc transplantation of LLC or inoculation sc of HBS. groups of l(}-l6 experimental (LLC) and control (HBS) mice were inoculated with C. 1l1bicIlns on each of the days indicated. • Mean survival after infection with C. Illbicllns ±8E. t Percent ratio of mean survival days for tumor·bearing/control mice. Significant increase in survival occurred at values ~ 140.
HBS
%
o
10 -'
3, 4
SUMMARY-Lethality of Candida albicans was monitored in (C57BL x DBA/2)F1 mice bearing the transplanted Lewis lung carcinoma and in sham-operated controls inoculated with Hanks' balanced salt solution_ The lethal response to C. albicans infection was significantly delayed in animals inoculated with the microorganism 6-16 days after transplantation of the Lewis lung carcinoma. Maximal increases in survival times were observed when C. albicans was inoculated 8-12 days following tumor transplantation. Therefore, a delay in the lethal response to C. albicans in this untreated model murine tumor system could be elicited through implantation of the Lewis lung carci· noma; preliminary studies with some other model murine tumors and with cell-free filtrates indicated that this phenom· enon is not restricted to the Lewis lung carcinoma.-J Natl Cancer Inst 55: 731-733, 1975.
The microorganism used throughout this investigation was C. albicans (ATCC #10261), maintained and cultured as previously described (12). Sterile 0.85% saline suspensions of washed yeastlike C. albicans preparations were adjusted to predetermined turbidity (#66 red filter) with a Klett-Summerson colorimeter. These cultures averaged 1 X 101 cells/m!. In all experiments 0.5 ml of the suspension (for control animals, 0.85% saline) was inoculated iv into a lateral tail vein of each mouse. Following inoculation with C. albicans the mice were observed daily and mortality rates recorded. Animals were routinely examined at death for gross pathology. Mouse mortality rates were evaluated statistically by the Wilcoxon method (13). RESULTS AND DISCUSSION
Opportunistic candidiasis occurs in cancer patients and in naturally or artificially immunosuppressed hosts (1-5). However, it is uncertain whether candidiasis results primarily from an intrinsic defect in host resistance caused by the cancer or from immunosuppression caused by anticancer therapy. These clinical problems have stimulated attempts to define the factors influencing host resistance to this microorganism (6-8). However, there have been no reports in the literature concerning host resistance to Candida albicans in a model tumor system. In the present study, we examined the response of mice bearing the Lewis lung carcinoma (LLC) to challenge with a lethal injection of C. albicans in the absence of antitumor or antimicrobial therapy. MATERIALS AND METHODS
(C57BL X DBA/2)Fl (BDFl) mice were obtained from The Jackson Laboratory, Bar Harbor, Maine. At the start of each experiment, the animals were 6 weeks old and weighed 17-22 g. The transplanted LLC originated from a spontaneous (anaplastic) carcinoma discovered in the lung of a C57BL mouse by Margaret R. Lewis in 1951 (9). This tumor, which metastasizes rapidly to the lung (10), was maintained and transplanted sc every 16-18 days in the syngeneic C57BL/6J mouse, as described by Mayo (11). All subsequent experiments were done with BDFI mice. A I-mm2 fragment of the LLC was implanted sc into the left hindquarter of each BDFI mouse receiving a tumor implant. Sham operations were done on anesthesized BDFI mice (controls), and these animals were then inoculated sc with 0.1 ml Hanks' balanced salt solution (HBS). In some experiments, shamoperated BDFI mice were inoculated sc with 0.1 ml MiIIipore filtrate (0.45 nm porosity) from minced LLC cells harvested 16-18 days after transplantation into C57BL/6J mice. This LLC filtrate (LLF) was prepared by aseptic suspension of 60 pieces of tumor (::::::1.0 mm 2 each) in 10 ml sterile HBS. The preparation was filtered (MiIlipore), and 0.1 ml was implanted sc into each mouse at the site of the incision. No mice inoculated with the filtrate developed tumors within 60 days.
A significant delay in the lethal response to C. albicans in BDFI mice bearing transplanted LLC occurred when mice were challenged with this microorganism 8 days after tumor implantation (text-fig. I). Conversely, when mice were infected with C. albicans 0, 2, or 4 days following implantation of LLC fragments, no significant delay in mortality was observed (table 1). Maximal increased survival times occurred when C. albicans was injected into mice 8-12 days after tumor implantation. Control mice bearing only the carcinoma began to die by day 18; therefore, reliable data could not be obtained beyond 16 days. Injection of C. albicans into BDFI mice 8 days after implantation of LLC fragments, other model murine tumors, or cell-free filtrates from the tumors (table 2) indicated that this phenomenon, though not universal, is not unique to the LLC system. Furthermore, these preliminary data suggest that a delayed lethal response similar to that observed with tumor fragments can be obtained with Millipore filtrates from tumor preparations. This effect was not seen when organ filtrates from normal BDFl mice were used in place of tumor filtrates. Thus experiments suggest that the activity against C. albicans does not require transplantation of tumor cells per se but depends on a component from the malignant cells. The availability of Millipore filtrates as suitable mediators of a delayed lethal response to C. albicans should allow us to extend our studies into the nature of the response to this microorganism elicited in the tumor-bearing host. Since the filtrates used in these experiments were prepared from tumor cells harvested only once after implantation, it is presently unknown whether material with greater activity can be prepared from tumor cell filtrates obtained at different intervals during carcinogenesis. Received April I, 1975; accepted May 30, 1975. Supported in part by Public Health Service grant CAI6627 from the National Cancer Institute and by grant ACS IN-105 from the American Cancer Society. 3 Department of Microbiology, Box 847, Medical College of Virginia, Virginia Commonwealth University, Richmond, Va. 23298. 4 We thank Ms. Trisha Reed for her technical assistance, and Drs. A. E. Munson, A. M. Kaplan, and P. S. Morahan for their helpful discussions. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.3, SEPTEMBER 1975
731
732
ROBINETTE AND MARDON
pared with LLC-bearing mice and those receiving LLF (text-fig. 2). 100
TABLE
2.-Mean survival times for mice inoculated with C. albicans
8 days after implantation with murine tumors and Millipore
filtrates from tumor preparations
80
Implant a
Survival (days) b
Tumor-bearing/ control c
60
Lewis lung carcinoma ___________ Millipore filtrate _______________ HBS controL _________________
7.3±OA 5.9±0.5 4.1±O.7
178 144 100
40
B-16 melanotic melanoma _______ Millipore filtrate _______________ HBS controL _________________
7.6±0.S 5.6±O.5 4.0±O.6
190 141 100
3-Methylcholanthrene-induced fibrosarcoma-l d Millipore filtrate _______________ HBS controL __________________
7A±0.6
20
186
6.S±0.6 4.0±0.6
170 100
6.6±0.7
124
5.7±0.4 5.3±0.4
106 100
III III:
0
>
>
III:
:::l
III ~
z
""U ""a..
III:
12
8
14
3-Methylcholanthrene-induced fibrosarcoma-2 d Millipore filtrate _______________ HBS controL _________________
16
DAYS POST IWOCULATION I.-Mortality from C. albicans in groups of mice bearing LLC or in groups of sham-operated control mice receiving HBS. Each animal was inoculated 8 days (vertical dotted line) after LLC or HBS implantation, with 0.5 ml standard cell suspension of yeastlike C. albicans. Curves represent data from 3 experiments, each consisting of experimental and control groups of 10 mice each. Variation among the 3'experiments: P:S;0.05.
TEXT-FIGURE
TABLE
I.-Mean survival times for mice inoculated with C. albicans at selected intervals following LLC implantation
Day post implantation a
0 2 4 6 8 10 12 14 16
Survival (days)
b
LLC
HBS
4.5±OA 5.3±O.8 4.8±0.8 6.3±O.8 7.3±0.4 8.7±1.0 7.8±0.6 8.2±1.1 6.8 ± 1.1
4.2±O.6 4.7±O.7 4.5±0.7 4.2±0.6 4.1±0.7 5.0±1.0 4A±0.4 5.0±1.0 4A±1.1
• All experimental and control BDFI mice received sc in the left hindquarter appropriate tumor fragments, filtrate from minced tumor cells prepared as described in "Materials and Methods." or HB8. Each value represents data from a group of 16 BDF, mice. All tumors were obtained from C57BL mice. Eight days after implantation, each mouse was challenged iv with 5 X 106 yeastIike cells of C.1l1bicans. • Mean survival after infection with C. albicllns ±SE. , Percent ratio of mean survival days for tumor-bearing/control mice. Significant increase in survival occured at values? 140. d 3-Methylcholanthrene-induced fibrosarcoma-l was maintained via multiple serial transplants in C57BL mice. 3-Methylcholanthrene-induced fibrosarcoma-2 was maintained frozen in liquid nitrogen and used after the first transplant into C57BL mice.
Tumor-bearing/ control c
15
107 113
107 150 180 174 177 164 154
• Following sc transplantation of LLC or inoculation sc of HBS. groups of l(}-l6 experimental (LLC) and control (HBS) mice were inoculated with C. 1l1bicIlns on each of the days indicated. • Mean survival after infection with C. Illbicllns ±8E. t Percent ratio of mean survival days for tumor·bearing/control mice. Significant increase in survival occurred at values ~ 140.
HBS
%
o
10 -'
