Postoperative
Immunotherapy
By Carol L.
of Murine C1300-Neuroblastoma
Fowler, Stephen P. Brooks, Jon E. Rossman, and Donald R. Cooney Buffalo,
New
0 Low-dose cyclophosphamide (CY) is an immunomodulating agent that down-regulates T suppressor cell function. This study investigates postoperative immunotherapy with CY as an alternate treatment for advanced immunogenic tumors such as neuroblastoma that typically respond poorly to traditional high-dose chemotherapy. A/J mice with 1.5cm subcutaneous Cl 3W-neuroblastoma (Cl 3WNB) tumors were divided into the following treatment groups: I, untreated (n = 14); II, 85% tumor resection (n = 18): Ill. sham-operated (n = 18); IV, multiple-dose CY (n = 6); V, 85% resection and single-dose CY (n = 14); VI, 85% resection and multiple-dose CY (n = 14). CY (100 mg/kg, intraperitoneally) was given initially 24 hours postoperatively to groups IV, V. and VI. Groups IV and VI also received weekly maintenance doses of 25 mg/kg CY. Results showed significantly increased survival (log-rank test) in CY-treated groups (IV, V, VI) compared with control groups ~1.11.111). Cures were observed only in groups receiving partial resection plus CY (V, 7%; VI, 29%). Although surgical debulking of tumor alone (II) did not enhance survival, the procedure normalized depressed total lymphocyte counts and the subpopulation of Lyt 2.3+ (T suppressor/cytolytic cells) in the immediate postoperative period during which immunotherapy with CY was instigated. This may have contributed to the success of CY immunotherapy. To characterize the tumor-host immune interaction, additional studies were performed. Results showed the following. (1) Mice cured by debulking plus CY (from groups V and VII could not be successfully reimplanted with Cl 3W-N8, demonstrating immunologic mediation by CY. (2) Injection of antithymocyte serum abrogated the therapeutic effects of CY in tumor-bearing mice, indicating that CY action is T cell-mediated. (3) T cell Concanavalin-A blastogenesis assay demonstrated suppression of T cell stimulation in tumor-bearing mice. This tumor-associated suppression was eradicated by CY treatment (188 mg/kg) of tumor-bearing mice. (4) Treatment of tumor-bearing mice with intravenous anti-IJx serum significantly delayed tumor growth (P = 882). demonstrating a role of IJK cells in C13W-N8 growth. These IJK cells are part of the T suppressor network. (5) An increased polymorphonuclear cell count in the peripheral blood was a marker for the presence of tumor tissue, even before the gross tumor was noted. Because this evidence suggests that tumor-associated T suppressor populations contribute to experimental neuroblastoma growth, a trial of treatment of neuroblastoma patients with low-dose CY to inactivate these populations and support host antitumor mechanisms may be indicated, especially in the face of treatment failures with high-dose CY. @ 1990 by W.B. Saunders Company. INDEX WORDS: Cyclophosphamide; immunotherapy; neuroblastoma.
T
HE CURRENT treatment of advanced neuroblastoma with high-dose chemotherapy and irradiation is unsatisfactory because it has not significantly Journal of Pediatric Surgery, Vol 25, No 2 (February), 1990: pp 229-237
York
increased overall patient survival in the past two decades.’ Furthermore, although palliation is occasionally achieved, it is usually accompanied by toxic side effects.2 A more efficacious and less toxic form of therapy for neuroblastoma is needed, and immunotherapy may provide such an alternative. Neuroblastoma evokes host immune responses both clinically and in experimental models.3*4Occasionally, spontaneous tumor regression occurs,’ and is possibly a consequence of the host immune response.6 Because these observations suggest that neuroblastoma is immunogenic, it has been postulated that immunotherapy might improve survival of affected patients by enhancing the host antitumor response.‘,* The syngeneic murine neuroblastoma, C 1300-NB, should provide an acceptable tumor model on which to test immunotherapies, because it has been shown previously that Cl 300-NB has immunogenic properties.2 While cyclophosphamide (CY) is traditionally used in high doses to treat neuroblastoma, there is evidence that in lower doses it is an immunomodulating agent that down-regulates T suppressor (Tsupp) cell function.‘-” This property of low-dose CY is potentially useful in tumor therapy because one mechanism by which tumors may evade the host antitumor response is to stimulate Tsupp function, which in turn inhibits the host antitumor effector cells.‘2*13Experimentally, preferential down-regulation of tumor-associated Tsupp cell function has mediated regression of established tumors in animal models.‘4*‘5 In this study, postoperative treatments using immunoregulating doses of CY in a clinically relevant model of incompletely resected, advanced murine neuroblastoma were evaluated. MATERIALS AND METHODS FemaleA/J mice, 8 to 10 weeks old, were obtained from Jackson
Laboratories (Bar Harbor, ME). C1300-NB has been maintained in From the Department of Pediatric Surgery, Children’s Hospital of Buflalo, University of Buffalo, and the State University of New York, Buffalo, NY. Supported in part by the Women & Children’s Health Research Foundation, Inc. Children’s Hospital, Buffalo, NY, and the James H. Cummings Foundation, Inc. Buffalo, NY. Presented at the 20th Annual Meeting of the American Pediatric Surgical Association, Baltimore. Maryland, May 28-31 I 1989. Address reprint requests to Donald R. Cooney, MD, Head, Department of Pediatric Surgery, ChildrenS Hospital of Buffnlo. 219 Bryant St, Buffalo, NY 14222. o 1990 by W.B. Saunders Company. 0022-3468/90/2502-0009$03.00/0
229
FOWUR ET AL
230 our laboratory by serial passage both in vivo and in vitro. Only in vivo passaged tumor was used in the present study. For passage in vivo, tumor cell suspensions were prepared by aseptically excising subcutaneous tumor tissue, then mechanically disaggregating it in phosphate-buffered saline (PBS) (pH, 7.2). Viability of cells was determined by trypan blue exclusion. A suspension of 1 x lo6 live tumor cells was then injected subcutaneously in the right flank.
Cyclophosphamide CY was obtained from Sigma Chemical Co (St Louis, MO). Each dose of CY was administered as a single intraperitoneal (IP) injection on the days indicated in the treatment protocol.
Surgical Procedure When tumors were 1.5 cm in diameter (10 days after the implantation), mice were anesthetized with sodium pentobarbital. Sharp resection of 85% of the tumor was performed under sterile conditions, and the wound was closed primarily.
Treatment Schedules Eighty-four mice with l&cm Cl300-NB tumors were divided into the following experimental groups (Fig 1): I, untreated controls (n = 14); II, operative controls, with 85% tumor resection (n = 18); III, sham-operated controls, with a skin incision and partial mobilization of the tumor but no resection (n = 18); IV, no tumor resection, and multiple-dose CY (n = 6); V, 85% tumor resection and single-dose CY (n = 14); VI, 85% tumor resection and multipledose CY (n = 14). CY was administered (100 mg/kg IP) to groups IV, V, and VI on day 11 (24 hours after surgery in operated groups V and VI). Mice in groups IV and VI also received low-maintenance doses of CY each week (25 mg/kg). Treatment was continued until 60 days after initial tumor implantation.
Immunologic Studies Lymphocyte phenotype studies. Postbulbar blood was collected from groups I to VI on day 10 (prior to surgery), day 13 (2 days after CY treatment, postoperative day 3 [POD 3]), then weekly until day 60. The blood collections were performed 2 days after CY treatment (each week). Lymphocyte subsets were labeled with the monoclonal antibodies antiLyt 2,3 (antisuppressor/cytolytic T cells) followed by
Day
0
10
I ,_ .
Cl300
I
I
Untreated
II
OP
I I Sham OP
IV
CY
v
OPtCYxl
VI
OPtCY/wk
11
..I ^, I f t op CY-100
FITC (second antibody), and antiIgG-FITC (anti-IgG+ B cells; all were obtained from Becton Dickinson, Mountain View, CA). Flow cytometric analysis was performed with Cytofluorograf, System 50-L (Ortho Instruments, Westwood, MA). Twelve to 24 normal mice without tumors served as controls for each time period. White blood cell (WBC) and differential counts. WBC counts by the standard Coulter counter method and differential counts from Wright-stained smears were performed concurrently with lymphocyte subset determinations in groups I to VI. Twelve to 24 mice served as normal controls. Reimplantation of tumor. All mice from groups I to VI noted to be cured of the tumor on day 60 after initial tumor implantation were observed for an additional 30 days with no further treatment. If no tumor regrowth was apparent, the animals were reimplanted with 1 x lo6 Cl300-NB cells. Mice were observed for another 60 days for reappearance of the tumor. Three comparison groups were formed, including group 1 with 1.5-cm tumors treated with very high-dose CY (300 mg/kg, n = 4). Group 2 animals, also with 1.5~cm tumors, were treated w?& 300 mg/kg CY one day after 85% tumor resection (n = 4). Group 3, with early 0.5-cm tumors, was treated by complete excision (n = 4). With no regrowth of neuroblastoma observed in 30 days, 1 x lo6 Cl 300-NB cells were reimplanted. Mice were examined daily for 60 days for signs of tumor growth. T cell Concanavalin A (Con-A) blastogenesis assay. Thirty-four mice were divided into three groups to test T cell suppressor function. Group 1 consisted of normal mice (n = 12); group 2 (n = 11) consisted of mice bearing 1.5-cm tumors; group 3 mice (n = 11) also had 1.5-cm tumors, but were treated with a single dose of CY (100 mg/kg). Suppressor function was tested 48 hours after CY adminisPooled peripheral blood (1 mL) from three mice in each group was obtained by retrobulbar puncture, diluted at a 1:2 ratio in Hanks balanced salt solution (HBSS) and applied to a gradient of Histopaque 1.083 (Sigma Chemical Co, St Louis, MO). Following centrifugation at 1,500g for 30 minutes at 25“C, the mononuclear cell layer was removed and washed once in HBSS and twice in RPM1 1640 media. The cell pellet was resuspended in 0.5-mL AIM-V serum-free media (GIBCO, Grand Island, NY) that was supplemented with 5 x 10e5 mol/L 2-mercaptoethanol. In duplicate, 3 x 10’ lymphocytes were diluted to a total volume of 0.5 mL
18
25
I
I
r
CY-25
t
CY-25
60
‘+
t
(+I
t
t
Fig 1. All groups were injected with C1300-NB (1 x 10’ cells) subcutaneously on day 0, then received various treatment regimens including partial tumor resection end cyclophosphemide starting on day 10. OP = 86% tumor resection; CY-100 = CY 100 mglkg IP: CY-25 = CY 25 mg/kg IP.
IMMUNOTHERAPY
FOR MURINE NEUROBLASTOMA
231
with supplemented AIM-V. One of the two tubes received 20 @/mL Con-A. Samples were incubated at 37OC in 5% CO, for 48 hours. Tritiated thymidine (0.05 microcuries per tube) was added to all tubes, and incubation continued for an additional 24 hours. The cells were washed three times in PBS, 0.25 mL of a 1:2 ratio Protosol (NEN-DuPont, Wilmington, DE)/ethanol solution was added, then 10 mL of Liquifluor scintillation fluid (NEN-Dupont, Wilmington, DE) was added for determination of tritiated thymidine incorporation with an LS-31331 Liquid Scintillation Counter (Beckman Instruments, Fullerton, CA). This procedure was performed in quadruplicate, and the results of stimulation indexes are expressed as group means + SE. The stimulation index (SI) was determined using the formula: SI =
mean experimental counts per minute (with Con-A) mean background counts per minute (without Con-A)
In vivo thymocyte depletion. Five mice with 0.75cm tumors were treated with CY (100 mg/kg) on day 8, then received antithymocyte serum (ATS; 0.25 mL IP) on days 10, 12, and 14, as in a protocol published by Dray and Mokyr.” Survival was compared with that of control groups that received ATS alone (n = 5) and CY alone (n = 5). In vivo inactivation of IJ’ cells. Four mice with l-cm (7-day) tumors were treated with anti-IJ’ serum (Accurate Chemical and Scientific Corp. Westbury, NY). Mice received 0.2 mL of antiserum daily by a tail vein injection on days 7.8, 10, and 13. Tumor growth was determined daily as the product of greatest width times length, and reported in square centimeters. The growth curves of untreated tumors were used for comparison.
Statistical
Analysis
of Data
Survival was followed for 60 days after tumor implantation, and groups were compared with untreated control group I using the log-rank test. P values less than or equal to .05 were considered statistically significant. Additionally, survival of groups was expressed as means + SD, but was included for gross comparison only. Other data were compared by ANOVA and Neuman-Keuls multiple comparison tests. RESULTS
Survival
There was no statistical difference in survival among the three control groups (I, no treatment; II, 85% tumor resection; III, sham operation) (Fig 2, Table 1). Mean survival in these groups was 23 to 24 days. Survival was significantly increased in all three groups receiving CY treatment (IV to VI). When resection was followed by a single postoperative dose of 100 mg/kg CY (group V), survival was significantly increased over that of controls (P c .OOl), and one (7%) tumor cure was noted. Survival was improved further with the addition of weekly 25mg/kg maintenance doses of CY (group VI, P < .OOl). Mean survival of this group increased to 51 f 9.9 days, and seven mice (50%) lived longer than 60 days. Four of these seven mice were cured of the tumor, and three were alive at 60 days with the tumor present. Groups IV and VI received identical multi-dose CY treatment; however, the tumor was not resected in
l
I
0 II
Untreated
A IV CY
OP
c v
6 III Sham OP
.
OP+CYx1
VI OP+CY/wk
Kaplan-Meier survival curves of treatment groups I to VI Fig 2. are shown. Pvalues (log-rank test) are shown in Table 1.
group IV. Surgical resection in group VI increased survival significantly (P < .05), with mean survival changing from 33 -I 2.6 days to 51 * 9.9 days. Cell Populations WBC and differential counts. WBC counts increased directly with tumor growth, from 6,152 2 1,714/mm3 in normal mice, up to 20,000 to 70,000/ mm3 in mice with very advanced tumors. As demonstrated by the differential counts, the leukocytosis reflected a proliferation of polymorphonuclear cells (PMNs), which increased from 23.5 rt 10.3% in normal mice to 80 to 95% of the leukocytes in mice with very advanced tumors (Fig 3). Elevation of the PMN count, as determined by either differential or absolute count, was a sensitive indicator of the presence of tumor and preceded the gross appearance of the tumor. The WBC and PMN responses were not accompanied by signs of infection or acute inflammation in the mice. Inversely related to the increasing number of PMNs in tumor-bearing mice was a simultaneous decrease in lymphocyte counts (Fig 3). The normal range for mice was 69.8 + 10.9%, but this diminished to about 5% in mice with very advanced tumors. Both B cell and Tsupp (Lyt 2,3+) cell counts were affected by the decreasing number of lymphocytes in mice with advanced tumors. For purposes of discussion, Lyt 2,3+ cells will be referred to as Tsupp cells, although it is
FOWLER ET AL
232
Table 1. Therapeutic
Groups
”
I Untreated II Operated Ill Sham-operated IV CY (100
+ 25/wk)
V Operation + CY ( 100) VI Operation + CY (100
+ 25/wk)
Results No.
Surviving at 60 Days
Survivalin Days (mean * SD)
No. of ClWS
14
23 f 2.3
0
0
18
24 ” 4.4, NS
0
0
18
23 +- 4.5, NS
6
33 f 2.6, P
Immunotherapy
By Carol L.
of Murine C1300-Neuroblastoma
Fowler, Stephen P. Brooks, Jon E. Rossman, and Donald R. Cooney Buffalo,
New
0 Low-dose cyclophosphamide (CY) is an immunomodulating agent that down-regulates T suppressor cell function. This study investigates postoperative immunotherapy with CY as an alternate treatment for advanced immunogenic tumors such as neuroblastoma that typically respond poorly to traditional high-dose chemotherapy. A/J mice with 1.5cm subcutaneous Cl 3W-neuroblastoma (Cl 3WNB) tumors were divided into the following treatment groups: I, untreated (n = 14); II, 85% tumor resection (n = 18): Ill. sham-operated (n = 18); IV, multiple-dose CY (n = 6); V, 85% resection and single-dose CY (n = 14); VI, 85% resection and multiple-dose CY (n = 14). CY (100 mg/kg, intraperitoneally) was given initially 24 hours postoperatively to groups IV, V. and VI. Groups IV and VI also received weekly maintenance doses of 25 mg/kg CY. Results showed significantly increased survival (log-rank test) in CY-treated groups (IV, V, VI) compared with control groups ~1.11.111). Cures were observed only in groups receiving partial resection plus CY (V, 7%; VI, 29%). Although surgical debulking of tumor alone (II) did not enhance survival, the procedure normalized depressed total lymphocyte counts and the subpopulation of Lyt 2.3+ (T suppressor/cytolytic cells) in the immediate postoperative period during which immunotherapy with CY was instigated. This may have contributed to the success of CY immunotherapy. To characterize the tumor-host immune interaction, additional studies were performed. Results showed the following. (1) Mice cured by debulking plus CY (from groups V and VII could not be successfully reimplanted with Cl 3W-N8, demonstrating immunologic mediation by CY. (2) Injection of antithymocyte serum abrogated the therapeutic effects of CY in tumor-bearing mice, indicating that CY action is T cell-mediated. (3) T cell Concanavalin-A blastogenesis assay demonstrated suppression of T cell stimulation in tumor-bearing mice. This tumor-associated suppression was eradicated by CY treatment (188 mg/kg) of tumor-bearing mice. (4) Treatment of tumor-bearing mice with intravenous anti-IJx serum significantly delayed tumor growth (P = 882). demonstrating a role of IJK cells in C13W-N8 growth. These IJK cells are part of the T suppressor network. (5) An increased polymorphonuclear cell count in the peripheral blood was a marker for the presence of tumor tissue, even before the gross tumor was noted. Because this evidence suggests that tumor-associated T suppressor populations contribute to experimental neuroblastoma growth, a trial of treatment of neuroblastoma patients with low-dose CY to inactivate these populations and support host antitumor mechanisms may be indicated, especially in the face of treatment failures with high-dose CY. @ 1990 by W.B. Saunders Company. INDEX WORDS: Cyclophosphamide; immunotherapy; neuroblastoma.
T
HE CURRENT treatment of advanced neuroblastoma with high-dose chemotherapy and irradiation is unsatisfactory because it has not significantly Journal of Pediatric Surgery, Vol 25, No 2 (February), 1990: pp 229-237
York
increased overall patient survival in the past two decades.’ Furthermore, although palliation is occasionally achieved, it is usually accompanied by toxic side effects.2 A more efficacious and less toxic form of therapy for neuroblastoma is needed, and immunotherapy may provide such an alternative. Neuroblastoma evokes host immune responses both clinically and in experimental models.3*4Occasionally, spontaneous tumor regression occurs,’ and is possibly a consequence of the host immune response.6 Because these observations suggest that neuroblastoma is immunogenic, it has been postulated that immunotherapy might improve survival of affected patients by enhancing the host antitumor response.‘,* The syngeneic murine neuroblastoma, C 1300-NB, should provide an acceptable tumor model on which to test immunotherapies, because it has been shown previously that Cl 300-NB has immunogenic properties.2 While cyclophosphamide (CY) is traditionally used in high doses to treat neuroblastoma, there is evidence that in lower doses it is an immunomodulating agent that down-regulates T suppressor (Tsupp) cell function.‘-” This property of low-dose CY is potentially useful in tumor therapy because one mechanism by which tumors may evade the host antitumor response is to stimulate Tsupp function, which in turn inhibits the host antitumor effector cells.‘2*13Experimentally, preferential down-regulation of tumor-associated Tsupp cell function has mediated regression of established tumors in animal models.‘4*‘5 In this study, postoperative treatments using immunoregulating doses of CY in a clinically relevant model of incompletely resected, advanced murine neuroblastoma were evaluated. MATERIALS AND METHODS FemaleA/J mice, 8 to 10 weeks old, were obtained from Jackson
Laboratories (Bar Harbor, ME). C1300-NB has been maintained in From the Department of Pediatric Surgery, Children’s Hospital of Buflalo, University of Buffalo, and the State University of New York, Buffalo, NY. Supported in part by the Women & Children’s Health Research Foundation, Inc. Children’s Hospital, Buffalo, NY, and the James H. Cummings Foundation, Inc. Buffalo, NY. Presented at the 20th Annual Meeting of the American Pediatric Surgical Association, Baltimore. Maryland, May 28-31 I 1989. Address reprint requests to Donald R. Cooney, MD, Head, Department of Pediatric Surgery, ChildrenS Hospital of Buffnlo. 219 Bryant St, Buffalo, NY 14222. o 1990 by W.B. Saunders Company. 0022-3468/90/2502-0009$03.00/0
229
FOWUR ET AL
230 our laboratory by serial passage both in vivo and in vitro. Only in vivo passaged tumor was used in the present study. For passage in vivo, tumor cell suspensions were prepared by aseptically excising subcutaneous tumor tissue, then mechanically disaggregating it in phosphate-buffered saline (PBS) (pH, 7.2). Viability of cells was determined by trypan blue exclusion. A suspension of 1 x lo6 live tumor cells was then injected subcutaneously in the right flank.
Cyclophosphamide CY was obtained from Sigma Chemical Co (St Louis, MO). Each dose of CY was administered as a single intraperitoneal (IP) injection on the days indicated in the treatment protocol.
Surgical Procedure When tumors were 1.5 cm in diameter (10 days after the implantation), mice were anesthetized with sodium pentobarbital. Sharp resection of 85% of the tumor was performed under sterile conditions, and the wound was closed primarily.
Treatment Schedules Eighty-four mice with l&cm Cl300-NB tumors were divided into the following experimental groups (Fig 1): I, untreated controls (n = 14); II, operative controls, with 85% tumor resection (n = 18); III, sham-operated controls, with a skin incision and partial mobilization of the tumor but no resection (n = 18); IV, no tumor resection, and multiple-dose CY (n = 6); V, 85% tumor resection and single-dose CY (n = 14); VI, 85% tumor resection and multipledose CY (n = 14). CY was administered (100 mg/kg IP) to groups IV, V, and VI on day 11 (24 hours after surgery in operated groups V and VI). Mice in groups IV and VI also received low-maintenance doses of CY each week (25 mg/kg). Treatment was continued until 60 days after initial tumor implantation.
Immunologic Studies Lymphocyte phenotype studies. Postbulbar blood was collected from groups I to VI on day 10 (prior to surgery), day 13 (2 days after CY treatment, postoperative day 3 [POD 3]), then weekly until day 60. The blood collections were performed 2 days after CY treatment (each week). Lymphocyte subsets were labeled with the monoclonal antibodies antiLyt 2,3 (antisuppressor/cytolytic T cells) followed by
Day
0
10
I ,_ .
Cl300
I
I
Untreated
II
OP
I I Sham OP
IV
CY
v
OPtCYxl
VI
OPtCY/wk
11
..I ^, I f t op CY-100
FITC (second antibody), and antiIgG-FITC (anti-IgG+ B cells; all were obtained from Becton Dickinson, Mountain View, CA). Flow cytometric analysis was performed with Cytofluorograf, System 50-L (Ortho Instruments, Westwood, MA). Twelve to 24 normal mice without tumors served as controls for each time period. White blood cell (WBC) and differential counts. WBC counts by the standard Coulter counter method and differential counts from Wright-stained smears were performed concurrently with lymphocyte subset determinations in groups I to VI. Twelve to 24 mice served as normal controls. Reimplantation of tumor. All mice from groups I to VI noted to be cured of the tumor on day 60 after initial tumor implantation were observed for an additional 30 days with no further treatment. If no tumor regrowth was apparent, the animals were reimplanted with 1 x lo6 Cl300-NB cells. Mice were observed for another 60 days for reappearance of the tumor. Three comparison groups were formed, including group 1 with 1.5-cm tumors treated with very high-dose CY (300 mg/kg, n = 4). Group 2 animals, also with 1.5~cm tumors, were treated w?& 300 mg/kg CY one day after 85% tumor resection (n = 4). Group 3, with early 0.5-cm tumors, was treated by complete excision (n = 4). With no regrowth of neuroblastoma observed in 30 days, 1 x lo6 Cl 300-NB cells were reimplanted. Mice were examined daily for 60 days for signs of tumor growth. T cell Concanavalin A (Con-A) blastogenesis assay. Thirty-four mice were divided into three groups to test T cell suppressor function. Group 1 consisted of normal mice (n = 12); group 2 (n = 11) consisted of mice bearing 1.5-cm tumors; group 3 mice (n = 11) also had 1.5-cm tumors, but were treated with a single dose of CY (100 mg/kg). Suppressor function was tested 48 hours after CY adminisPooled peripheral blood (1 mL) from three mice in each group was obtained by retrobulbar puncture, diluted at a 1:2 ratio in Hanks balanced salt solution (HBSS) and applied to a gradient of Histopaque 1.083 (Sigma Chemical Co, St Louis, MO). Following centrifugation at 1,500g for 30 minutes at 25“C, the mononuclear cell layer was removed and washed once in HBSS and twice in RPM1 1640 media. The cell pellet was resuspended in 0.5-mL AIM-V serum-free media (GIBCO, Grand Island, NY) that was supplemented with 5 x 10e5 mol/L 2-mercaptoethanol. In duplicate, 3 x 10’ lymphocytes were diluted to a total volume of 0.5 mL
18
25
I
I
r
CY-25
t
CY-25
60
‘+
t
(+I
t
t
Fig 1. All groups were injected with C1300-NB (1 x 10’ cells) subcutaneously on day 0, then received various treatment regimens including partial tumor resection end cyclophosphemide starting on day 10. OP = 86% tumor resection; CY-100 = CY 100 mglkg IP: CY-25 = CY 25 mg/kg IP.
IMMUNOTHERAPY
FOR MURINE NEUROBLASTOMA
231
with supplemented AIM-V. One of the two tubes received 20 @/mL Con-A. Samples were incubated at 37OC in 5% CO, for 48 hours. Tritiated thymidine (0.05 microcuries per tube) was added to all tubes, and incubation continued for an additional 24 hours. The cells were washed three times in PBS, 0.25 mL of a 1:2 ratio Protosol (NEN-DuPont, Wilmington, DE)/ethanol solution was added, then 10 mL of Liquifluor scintillation fluid (NEN-Dupont, Wilmington, DE) was added for determination of tritiated thymidine incorporation with an LS-31331 Liquid Scintillation Counter (Beckman Instruments, Fullerton, CA). This procedure was performed in quadruplicate, and the results of stimulation indexes are expressed as group means + SE. The stimulation index (SI) was determined using the formula: SI =
mean experimental counts per minute (with Con-A) mean background counts per minute (without Con-A)
In vivo thymocyte depletion. Five mice with 0.75cm tumors were treated with CY (100 mg/kg) on day 8, then received antithymocyte serum (ATS; 0.25 mL IP) on days 10, 12, and 14, as in a protocol published by Dray and Mokyr.” Survival was compared with that of control groups that received ATS alone (n = 5) and CY alone (n = 5). In vivo inactivation of IJ’ cells. Four mice with l-cm (7-day) tumors were treated with anti-IJ’ serum (Accurate Chemical and Scientific Corp. Westbury, NY). Mice received 0.2 mL of antiserum daily by a tail vein injection on days 7.8, 10, and 13. Tumor growth was determined daily as the product of greatest width times length, and reported in square centimeters. The growth curves of untreated tumors were used for comparison.
Statistical
Analysis
of Data
Survival was followed for 60 days after tumor implantation, and groups were compared with untreated control group I using the log-rank test. P values less than or equal to .05 were considered statistically significant. Additionally, survival of groups was expressed as means + SD, but was included for gross comparison only. Other data were compared by ANOVA and Neuman-Keuls multiple comparison tests. RESULTS
Survival
There was no statistical difference in survival among the three control groups (I, no treatment; II, 85% tumor resection; III, sham operation) (Fig 2, Table 1). Mean survival in these groups was 23 to 24 days. Survival was significantly increased in all three groups receiving CY treatment (IV to VI). When resection was followed by a single postoperative dose of 100 mg/kg CY (group V), survival was significantly increased over that of controls (P c .OOl), and one (7%) tumor cure was noted. Survival was improved further with the addition of weekly 25mg/kg maintenance doses of CY (group VI, P < .OOl). Mean survival of this group increased to 51 f 9.9 days, and seven mice (50%) lived longer than 60 days. Four of these seven mice were cured of the tumor, and three were alive at 60 days with the tumor present. Groups IV and VI received identical multi-dose CY treatment; however, the tumor was not resected in
l
I
0 II
Untreated
A IV CY
OP
c v
6 III Sham OP
.
OP+CYx1
VI OP+CY/wk
Kaplan-Meier survival curves of treatment groups I to VI Fig 2. are shown. Pvalues (log-rank test) are shown in Table 1.
group IV. Surgical resection in group VI increased survival significantly (P < .05), with mean survival changing from 33 -I 2.6 days to 51 * 9.9 days. Cell Populations WBC and differential counts. WBC counts increased directly with tumor growth, from 6,152 2 1,714/mm3 in normal mice, up to 20,000 to 70,000/ mm3 in mice with very advanced tumors. As demonstrated by the differential counts, the leukocytosis reflected a proliferation of polymorphonuclear cells (PMNs), which increased from 23.5 rt 10.3% in normal mice to 80 to 95% of the leukocytes in mice with very advanced tumors (Fig 3). Elevation of the PMN count, as determined by either differential or absolute count, was a sensitive indicator of the presence of tumor and preceded the gross appearance of the tumor. The WBC and PMN responses were not accompanied by signs of infection or acute inflammation in the mice. Inversely related to the increasing number of PMNs in tumor-bearing mice was a simultaneous decrease in lymphocyte counts (Fig 3). The normal range for mice was 69.8 + 10.9%, but this diminished to about 5% in mice with very advanced tumors. Both B cell and Tsupp (Lyt 2,3+) cell counts were affected by the decreasing number of lymphocytes in mice with advanced tumors. For purposes of discussion, Lyt 2,3+ cells will be referred to as Tsupp cells, although it is
FOWLER ET AL
232
Table 1. Therapeutic
Groups
”
I Untreated II Operated Ill Sham-operated IV CY (100
+ 25/wk)
V Operation + CY ( 100) VI Operation + CY (100
+ 25/wk)
Results No.
Surviving at 60 Days
Survivalin Days (mean * SD)
No. of ClWS
14
23 f 2.3
0
0
18
24 ” 4.4, NS
0
0
18
23 +- 4.5, NS
6
33 f 2.6, P
