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Effect of Perioperative Chemoimmunotherapy with Cyclophosphamide and Autologous Tumor Vaccine in Murine MBT-2 Bladder Cancer
0022-5347 /94/1516-1680$03.00/0 THE JOURNAL OF UROLOGY Copyright© 1994 by AMERICAN UROLOGICAL ASSOCIATION, INC.
Vol. 151, 1680-1686, June 1994 Printed in U.S. A.
EFFECT OF PERIOPERATIVE CHEMOIMMUNOTHERAPY WITH CYCLOPHOSPHAMIDE AND AUTOLOGOUS TUMOR VACCINE IN MURINE MBT-2 BLADDER CANCER TZONG-SHIN TZAI, ROBERT P. RUBEN, GINTARAS ZALESKIS, ERICA S. BERLETH, M. JANE EHRKE* AND ENRICO MIRICH From the Department of Urologic Oncology and Grace Cancer, Drug Center, Roswell Park Cancer Institute, Buffalo, New York
ABSTRACT
The in vitro cytotoxic activity of splenocytes from C3H/He mice implanted subcutaneously with 106 syngeneic MBT-2 tumor cells on day O was significantly enhanced after cyclophosphamide (100 mg./kg., intraperitoneally) given 2 days before tumor resection on day 17, with or without active specific immunization with BCG plus autologous irradiated tumor cells (vaccine) 1 week after tumor resection. Furthermore, a significantly lower tumor incidence was seen in mice challenged with 105, but not 106 , tumor cells per mouse 24 hours after tumor resection on day 17 and treated with cyclophosphamide on day 15 and postoperatively with vaccine than was found in nontreated tumor resected mice. Phenotypic analysis of cells from spleen showed that cyclophosphamide pretreatment and postoperative vaccine, either singly or in combination, induced a significant increase of both CD44+ memory T cells and CDllb+ myeloid/macrophage cells. Thus, in addition to a specific antitumor immune response, a nonspecific cytolytic mechanism may also play a role in the observed antitumor effect. KEY WORDS: drug therapy, immunotherapy, cyclophosphamide
Treatment of moderate to advanced stages of cancer by surgery alone usually fails because of local recurrence and/or distant metastasis. The major reasons for the failure are believed to be: (1) tumor cell dissemination into the circulation during surgical manipulation;1· 2 (2) undetected residual local tumor or systemic micrometastasis of tumor; (3) further deterioration of the already suppressed host antitumor immune defenses due to surgical and/or anesthetic stress. 3- 5 Theoretically, immune stimulation immediately before or after tumor resection should reduce the chance for any residual tumor cells to grow. Cyclophosphamide (CY), when administered at an appropriately low dose, has been shown to have immunoaugmenting effects by being relatively toxic to the precursors of suppressor T cells, while sparing cytotoxic T lymphocyte precursor cells. 6 • 7 Active specific immunization, using a vaccine prepared by irradiated autologous tumor cells mixed with Bacillus CalmetteGuerin (BCG), has been demonstrated to induce specific antitumor responses in tumor-bearing animals 8 • 9 and to improve both disease-free interval and survival of patients with colorectal cancer and melanoma. 10• 11 In this study the C3H/He-MBT2 syngeneic murine bladder tumor model was used. The situation of bladder cancer patients treated with surgery was mimicked, and the perioperative immunomodulating effects of CY pretreatment, with or without postoperative tumor vaccine (TV) immunization, were studied. MATERIALS AND METHODS
Animals. Female C3H/HeJCr mice, 4 weeks old, were obtained from the Mammalian Genetics and Animal Production Section, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland. Mice received free access to mouse chow and water and were studied between 6 and 8 weeks of age. Tumor cell lines. The MBT-2 murine transitional cell carciAccepted for publication December 10, 1993. * Requests for reprints: Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263. This work was supported in part by grants CA15142 and CA16056 from the National Cancer Institute.
noma was originally induced by N-(4-(Nitro-2-furyl)-2-thiazolyl) formamide (FANFT) administration to C3H/He mice. 12 Cells from this tumor line were maintained in RPMI 1640 plus 10% fetal calf serum (FCS). Media and reagents. RPMI 1640 supplemented with 10% heat inactivated FCS (Hyclone Laboratories, Logan, Utah), 1 mM. sodium pyruvate, 0.1 mM. nonessential amino acids, 25 mM. Hepes, gentamicin 50 ,ug./ml., 2.0 mM. glutamine and 50 ,uM. 2-mercaptoethanol is termed complete medium. The endotoxin levels in the FCS were in the range of 0.005 to 0.033 ng./ ml. Cyclophosphamide (Sigma Chemical Co., St. Louis, Missouri, Lot No. 19F02541) was dissolved in 0.9% saline to a concentration of 10 mg./ml. Recombinant human interleukin2 (IL-2) was a gift from E.I. DuPont de Nemours Co., Inc., Glenolden, Pennsylvania (Lot No. 0024C). Based on published information and as confirmed in this laboratory by a standard bioassay, 1 DuPont unit equals 30 BRMP units, and the specific activity of this recombinant material is 3700 BRMP units per ,ug. protein. Bacillus Calmette-Guerin (BCG) (Tokyo strain, Japan BCG Co., Tokyo, Japan) was suspended in 0.9% saline at a concentration of 10 mg./ml. The BCG (1 mg. in 0.1 ml. saline) was mixed with 106 irradiated (60 Gy) MBT-2 cells (IRMBT-2) as the TV and was injected subcutaneously into the abdominal wall of the mice. MBT-2 cells which had received 60 Gy x-irradiation failed to develop tumors in naive mice and did not proliferate in culture. Therefore, this is considered a lethal dose. Tumor cell implantation. MBT-2 tumor cells, harvested from culture flasks by trypsinization (Trypsin 0.25%, Gibco, Grand Island, New York), were washed two times in complete medium, counted (Trypan blue exclusion) and diluted to 10 7/ml. in phosphate buffered saline (PBS). A 0.1 ml. aliquot was implanted subcutaneously into the lower abdominal area of each mouse. Sublethal whole body x-irradiation. A Siemens 250 KV X-ray unit was used. Mice were placed into a cylindrical Plexiglas restrainer in the axis of the X-ray beam. The skin-to-source distance was 47.5 cm., and the dose rate was 1.15 Gy per minute in air. The x-irradiated mice received a single nonlethal dose of 4 Gy on the day of tumor implantation.
1680
1681
PER!OPERATIVE CHEMOYMMUNOTHERAPY IN MURINE BLADDER CANCER
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Tumor inoculatioo
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A1110 = = = = = = = = = = I = = = = = = = = = = = = = = = = = = I N , A2 I 10 = = = = = = = = = = I = = = = = V = = = = = = = = = = = = I N , A3111 , T = = = = = = R = I IN, A4 I 11 , T = = = = = = = R = I
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B3111, T=======R= I B4! 11 , T =======R = I B519 , T = C Y = R = I B6! 9 , T = C Y = R = I
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FIG. 1. Schemes of treatment protocols of in vivo tumor challenge studies. T: 106 tumor cells subcutaneous!¥ inoculated on day O; CY: Cyclophosphamide, 100 mg./kg., intraperitoneally on day 15; R: Resection of tumor on day 17; I: Implantation of 10 (Gr.A) or 105 (Gr.Bl viable MBT-2 tumor cells subcutaneously on day 18; V: Tumor vaccine subcutaneous injection on day 24; CTL: Cytotoxic T-lymphocyte assay; PH: Phenotyping for immune memory cells: co4+44+, co3+44+ and CD8+nb+. IN: Tumor incidence 60 days after reimplantation on day 18. Individual panels describe experimental design and number of mice per group for experiments yielding data shown as follows: Panel A for figs. 2 and 3; Panel B for figs. 4, 6, and 7; Panel C for fig. 5 and table 2.
TABLE
1. Effect of a sublethal dose (4 Gy) whole body irradiation on
No. of Tumor Cells Inoculated 10 105 10• 103
102 10 1
8/12 3/12 2/12 3/12 3/12 3/12
= 72) (66.7%) (25.0%) (16.7%) (25.0%) (25.0%) (25.0%)
Irradiated (n = 72) 12/12 12/12 10/12 7/12 8/12 8/12
(100.0%) (100.0%) (83.3%) (58.3%) (66.7%) (66.7%)
CY(+) IRMBT-2(-) " CY(-) IRMBT-2(+) ,w CY(+) IRMBT-2( +)
**
@
35
P" value (n
6
MBT-2
o CY(-) IRMBT-2(-)
Percent Tumor Incidence by Day 45 Nonirradiated
Target
40
the tumor incidence
*
Q) [/]
0.0466 0.0002 0.0017 0.1069 0.0498 0.0498
"Fisher's exact test; p value: irradiated versus nonirradiated.
/Cl
30
Q.) Q.)
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u'"' lD
**
p< 0.01
*
p< 0.05
25 20
()
:;::
Tumorigenicity and immunogenicity study of MBT-2 in C3H/ HeJCr mice. One hundred and forty-four mice were randomly allocated to two groups. One group received 4 Gy of sublethal whole body x-irradiation immediately before tumor cell implantation. The two groups (x-irradiated and nonirradiated) were then divided into 6 subgroups (12 mice per subgroup) which were implanted with 106 , 105, 104 , 103 , 10 2 , 101 MBT-2 cells per mouse. Tumor growth was monitored twice a week. Surgical tumor resection and treatment with CY with or without TV. Mice implanted with 106 MBT-2 tumor cells on day O were divided into 5 groups: one group [tumor bearing mice (TBM)] received no further treatment; 2 groups received CY (100 mg./kg. intraperitoneally) treatment on day 15 and 2 groups received tumor vaccine on day 24. All groups except the TBM group underwent tumor resection on day 17. Ketamine
() Q.)
15
Cl, [/"J
~
10 5
12/1
25/1
50/1
100/1
E/T
FIG. 2. Effect of CY pretreatment and/or IRMBT-2 on specific cytotoxicity of splenocytes from day 17 TBM. Splenocytes (5 x 106/ ml.) obtained on day 17 from CY (100 mg./kg., intraperitoneally, day 15) treated or nontreated TBM (experimental design shown in fig. 1, A) were cocultured in presence of IL-2 (0.5 U/ml.) with/without IRMBT-2 (R/S ratio: 100/1) for 5 days. Assayed in 4 hour 51 Cr release assay. Data obtained at four E:T ratios shown.
1682
PERIOPERATIVE CHEMOIMMUNOTHERAPY IN MURINE BLADDER CANCER 50
TABLE
E/T 100/1 Target: MBT-2 Effector: Splenocytes
(l)
~
Gr'
40
2. In vivo tumor challenge study
Treatmenth
* P< 0.05
(l)
.;
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0::
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20
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(l)
0. rt)
Al A2 A3 A4 A5 A6
Control' TV" TR TR+TV CY+TR CY+TR+ TV
Bl B2 B3 B4 B5 B6
Control' TV' TR TR+TV CY+TR CY+TR+TV
~ 10
D2-S
D3-S
D4-S
FIG. 3. Suppression of specific cytolytic activity of splenocytes by fresh or cultured irradiated MBT-2 tumor cells and their conditioned medium. Splenocytes (5 X 106/ml.) pooled from 3 to 4 day-17 TBM (experimental design was as shown on first line of fig. 1, A) were cultured in all cases with 0.5 U IL-2/ml. for 5 days plus: (1) complete medium alone (control); (2) fresh IRMBT-2 at R:S ratio of 100/1 (IRMBT-2); (3) cellular component recovered after IRMBT-2 (5 X 103 cells/0.2 ml./well) were cultured for 5 days with daily change of medium (D5-C); supernatant harvested on days 1, 2, 3 and 4 from wells of cultured IRMBT-2 (Dl-S, D2-S, D3-S, D4-S); final concentrations in wells were 25% or 50% of total volume.
LN
Spleen (l)
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co
70
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OJ 60
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Target , MBT- 2
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40
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8/10 9/10 2/11 2/11 1/9 1/9
(80.0%) (90.0%) (18.2%) (18.2%) (11.1 %) (11.1%)
0.5 0.0073 0.0073 0.0045 0.0045
(50.0%) (10.0%) (36.4%) (9.1%) (11.1 %) (0.0%)
0.0704 0.4250 0.055 0.0913 0.0217
105 cells
Dl-S
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p• value (vs. control)
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Tumor Incidence 60 days after MBT-2 reimplantation on Day 18'
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FIG. 4. In vitro cytotoxicity assay of effector cells from spleen and LNs. Lymphocytes obtained on day 31 from spleen and bilateral superficial inguinal LN were cultured in presence of IL-2 (0.5 U/ml.) for 5 days. Five experimental groups (experimental design and number of mice per group shown in fig. 1, B) were (1) day-31 TBM; (2) TR: tumor resected on day 17; (3) TR+TV: TR followed 1 week later by tumor vaccine (TV) immunization; (4) CY+TR: CY 100 mg./kg. intraperitoneally given 2 days before TR; (5) CY +TR+TV: mice received all three treatments. This experiment has been repeated 4 times with similar results.
(2.5 mg./kg.) intraperitoneally was used as the general anesthetic during surgery. On day 31 the mice in the 5 groups and age-matched naive mice were sacrificed to assess the effector functions of spleen and lymph node cells ex vivo. Preparation of single cell suspensions from spleen and lymph nodes. Spleen and bilateral superficial inguinal lymph nodes (LN) were removed aseptically on the day of sacrifice. Single cell suspensions were prepared as described previously. 13 Generation and assay of cytolytic effector cells. Aliquots (0.1 ml.) of spleen and lymph node cells (5 x 106 /ml. complete medium) were added to six wells of a 96-well round bottom culture plate. Aliquots (0.1 ml.) of rIL-2 (1.0 U/ml.) were added to each well giving a final concentration of0.5 U/ml. The plates were incubated for 5 days. The cultured cells were then serially
5/10 1/10 4/11 1/11 1/9 0/9
'The experimental design and numbers of mice per group are shown in figure lC. Mice in groups A3-A6 and B3-B6 received 106 MBT-2 tumor cells, subcutaneously, on day 0. h CY: cyclophosphamide (100 mg.jkg. intraperitoneally on day 15). TR: Tumor resection on day 17. TV: tumor vaccine (BCG + 106 IRMBT-2) immunization on Day 24. ' All mice in the two sets of 6 subgroups received subcutaneous inoculation with viable MBT-2 tumor cells [10 6 cells (Gr.A) or 105 cells (Gr.BJ, respectively] on day 18 (24 hours after tumor resection). • Fisher's exact test. • Naive mice (did not receive tumor on day 0).
diluted (2-fold) into wells containing complete medium only, and the cytolytic activity was assessed in a standard 4 hour 51 Cr-release assay described elsewhere. 3 • 13 In certain experiments, in addition to the IL-2, IRMBT-2 stimulator cells (S) were added to the spleen and LN responder cells (R) at a final R:S ratio of 100 to 1. Quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) assay for transforming growth factor-beta (TGF-(3). The assay involves a competitive template technique for quantitation and has been recently described. 14 It is based on the technique reported by Dolnick et al. 15 Briefly, a known amount of internal standard, which competes identically for all reactants, is added to cellular RNA and subjected to RT/PCR. The standard is engineered to contain a unique restriction enzyme site. Therefore, following amplification and digestion, it is possible to determine the amount of the product derived from cellular versus standard RNA. Phenotyping of the splenocytes and LN cells: a three-color analysis. Aliquots of cells were stained for simultaneous threecolor analysis on a FACScan flow cytometer (Becton-Dickinson, San Jose, California). The following monoclonal antibodies were used: FITC labeled anti-CDllb (Mac-1), PE conjugated anti-CD4 and anti-CDS, biotinylated anti-CDS (all purchased from Caltag, San Francisco, California) and FITC labeled antiCD44 (Pharmingen, San Diego, California). Streptavidin-Red 670 (Gibco) was added to visualize biotinylated anti-CDS as a second step. Ten thousand events were acquired in list mode. Lysys II and Paint-A-Gate software (Becton-Dickinson) were used for subset evaluation. In vivo tumor challenge study. The experimental design is outlined schematically in figure 1, C. Eight mice with palpable tumors, which were induced by subcutaneous implantation of 106 viable MBT-2 tumor cells on day 0, had their tumors resected on day 17. Thirty-six of them had been pretreated with CY (100 mg./kg., intraperitoneally) two days (day 15) before tumor resection. For comparative purposes, these 36 CY treated and the 44 untreated mice, together with 40 naive mice, were randomized into parallel subgroups as shown in the scheme in figure 1, C. All mice were challenged by subcutaneous implantation of either 106 (six "A" groups) or 105 (six "B" groups) viable tumor cells per mouse on day 18 (24 hours after
PERIOPERP/f'!VE CHErviOHvllV'.fUl";OTHERAPY IN J\l!URH\JE BLADDER CANCER 1.100
1683
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0.700 0.600
Median Survival Time
<..
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0.500
Al: 50 Days
~
L :,
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A2: 37 Days
(A6)
0.400
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(A5)
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0.200
TR
(A4) (A3)
Control >TV (A2l Contro I
0.100
(Al)
0
30
20
10
0
50
40
60
Days
FIG. 5. Percentage of actuarial survival of in vivo tumor challenge study. Results shown were obtained with animals in groups Al through A6 of table 2. Experimental design and number of mice per group are shown in fig. 1, C.
CDS+ Naive Mice
D31 TBM
TR
TR+N CY+TR
lli!lil I
lil!iillli
-
CD4+ lllllllli'I
CD44+
LJ
CD44-
I
I
I
~ I
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15
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Percentage FIG. 6. Expression of CD44 on CD4+ and CDs+ T lymphocytes in spleen on day 31. Experimental conditions and number per mice per group are described in fig. 1, B. Percentage of CD44 expressing CD4+ and CDs+ T lymphocytes in spleens of mice in different groups, as determined by three-color fl.ow cytometry on day 31, are shown.
tumor resection) at a site adjacent to the surgical wound. Six days later (day 24) one-half of the mice received a subcutaneous injection of TV as indicated. Tumor outgrowth and survival of mice were monitored daily. Statistical analysis. Independent Student's t test and Fisher's Exact test using SPSSPC+ software were used for data analysis. RESULTS
Tumorigenicity and immunogenicity of MBT-2 tumor cells. Initial studies had indicated that, following subcutaneous injection of 10 6 MBT-2 cells, only 60 to 70% of C3H/HeJCr mice developed progressive disease. This low tumor incidence suggested that host antitumor defenses might be involved. An experiment was carried out, therefore, comparing tumor incidence in nonirradiated mice to that in immunosuppressed, lowdose, whole body irradiated mice to determine the possible contribution of host antitumor defense to the low tumor incidence seen. The number of tumor cells inoculated was varied
logarithmically from 10 1 to 106 , and the percentage of tumor incidence observed by day 45 was calculated for both the whole body irradiated (4 Gy) and nonirradiated groups. It is clear from table 1 that a) for both groups, the percent tumor incidence increased in a dose-responsive manner with inoculum size and that b) the dose-response curve for the irradiated group lies to the right of that for the nonirradiated group. Thus, the immunologically deprived animals were clearly more susceptible to the challenge of inoculated tumor cells throughout the range of inocula tested. Separate tests of statistical significance (Fisher's Exact Test) comparing the nonirradiated versus irradiated groups for each inoculum size confirmed the overall trend. Only one separate analysis, that for 103 inoculated cells, missed being significant at the 0.05 level. Since low-dose, whole body x-ray irradiation is known to temporarily ablate the host's specific antitumor immunity, but to have much less effect on macrophage/monocyte mediated defenses, the results obtained suggest that the MBT-2 tumor is immunogenic in the syngeneic C3H/HeJCr host.
1684
PERIOPERATIVE CHEMOIMMUNOTHERAPY IN MURINE BLADDER CANCER
Naive Mice
D31 TBM
TR TR+TV CY+TR CY+ TR+ TV
-
J
Spleen
LJLN
~ J
J
LJ _J
0
5
10
'
'
'
'
15
20
25
30
35
40
Percentage FIG. 7. Percentage of CD11b+ cells in spleen and LNs on day 31. Experimental conditions and number of mice per group are as described in fig. 1, B.
The x-irradiated MBT-2 tumor cells remain metabolically active and immunosuppressive for at least 5 days. One approach used to demonstrate that a host is mounting a specific response to a syngeneic tumor is to challenge lymphocytes from TBM in culture with metabolically inactivated (x-irradiated) tumor cells and assess the ability of these lymphocytes to lyse syngeneic tumor target cells. The addition of fresh x-irradiated (60 Gy) MBT-2 cells (IRMBT-2), to in vitro response cultures with splenocytes from untreated day-17 TBM (fig. 2), however, was found to significantly reduce the level of cytolytic activity that developed. The administration of a dose of CY, which has been reported6 to be immunoaugmenting, significantly elevated the level of lytic activity attained with or without the addition of x-irradiated tumor. In each case, however, the levels attained in the presence of x-irradiated tumor were lower than those in the corresponding culture without its addition, suggesting that the tumor was immunosuppressive. Since this finding appeared to be in disagreement with the in vivo tumor incidence findings, further experiments were carried out. In these experiments it was found that the cellular component of day-5 cultured IRMBT-2, or supernatants harvested daily from the same cultured IRMBT-2 cells, when added at the beginning of in vitro response cultures with splenocytes from day-17 TBM, all suppressed the generation of specific cytotoxic activity (fig. 3). In a separate experiment, it was found that 24 hour supernatants from nonirradiated MBT-2 cells were also suppressive but markedly less so than those from x-irradiated cells (data not shown). Based on the above findings, the considerable literature indicating a role for (TGF-/n in tumor-induced suppression, and the fact that elevated mRNA expression and protein production have been shown to be associated, 16 quantitative RT/ PCR for TGF-/3 mRNA was carried out with RNA extracted from nonirradiated and x-irradiated MBT-2 cells. No substantial difference was seen in the level of message between the two conditions. Studies to identify the soluble factor are continuing. Effect of CY pretreatment with or without postoperative TV immunization on the antitumor activity of splenocytes and LN cells from TBM. An alternative approach to demonstrate specific host responses is to examine the ability of cells from TBM undergoing curative treatments to lyse syngeneic tumor target cells. As shown in fig. 4, the specific cytolytic activity of effector cells generated from both spleen and tumor draining LN obtained from TBM sacrificed on day 31 was increased significantly (p <0.001 and p <0.01, respectively) when compared with that of cells from control (naive, nontumor bearing) mice.
They both decreased markedly (p <0.001, p <0.01) in the group which had undergone tumor resection (TR) two weeks (day 17) earlier. Relative to TR alone, the preoperative (day 15) administration of CY followed by postoperative (day 24) TV immunization significantly enhanced the antitumor activity of effector cells in spleen (p <0.001) but not in LN (p >0.05). Similarly, relative to the TR alone, the adjuvant postoperative immunization with TV also induced a significant augmentation of the antitumor activity of splenocytes (p <0.001) but not of LN cells (p >0.05). In contrast, when the effect of preoperative CY treatment was assayed at this point (day 31), 2 weeks after tumor resection and 16 days after CY injection, there was no difference in spleen (p >0.05) or LN (p >0.5) cells relative to those from the TR alone groups. However, when assessed 2 (day-17 TBM, fig. 2) or 6 days (day-21 TBM, data not shown) after CY treatment, augmentation was seen. In vivo tumor challenge study. In the animal model under study surgical resection of the tumor was complete in the large majority of cases. Since this is not always the case clinically, a technique was chosen to mimic the situation of possible residual tumor due to incomplete surgical removal of a primary bladder cancer or the presence of micrometastases at the time of surgery. The effect of the perioperative treatments on host specific antitumor immunity was examined by subcutaneous reimplantation of either 106 or 105 tumor cells per mouse 24 hours after surgical removal of the primary tumor as outlined in figure l,C. It was found 60 days later that tumor incidence in the control groups was greater following implantation with 106 cells (Al group) than with 105 cells (Bl group, 5 of 10 or 50% versus 8 of 10 or 80%). This means that the control group to which all treatment groups are compared is smaller for those receiving 105 cells than for those receiving 106 cells. For the mice receiving 106 cells the four (A3-6) treatment groups (day-17 TR, TR+TV, CY +TR and CY +TR+TV) all demonstrated augmented (p <0.01, versus control) antitumor activity (table 2). There was no statistical difference, however, between the four groups in terms of tumor incidence or survival times (table 2 and fig. 5). In groups of mice challenged with 105 tumor cells per mouse, TV immunization caused suppression of tumor growth in both naive mice [from 50% (Bl) to 10% (B2)] and tumor-resected mice [from 36.4% (B3) to 9.1% (B4)]. However, the differences were not statistically significant [p >0.05 (Bl versus B2), p >0.1 (B3 versus B4)]. The lack of clear statistical significance for treatments of groups B3-B5 was due to the low incidence of tumor in naive mice when implanted with 105 MBT-2 tumor cells per mouse, since the outcome of treatment in these groups did not differ from that seen in groups A3-A5. Nevertheless, the triple treatment (group B6) was significantly better (p <0.025) than control, and the fact that not a single mouse in group B6 had tumor growth indicates the therapeutic efficacy of combined therapy of CY pretreatment and postoperative TV immunization with minimal residual tumor burden (10 5 cells) after surgery. Phenotypic analysis of leucocyte subsets. If specific host defense mechanisms were involved in the overall efficacy of the combination treatments, then one would expect development of memory T cells. To determine if the effect of CY pretreatment with or without postoperative TV immunization resulted in phenotypically definable memory subsets in spleen and/or LN of treated mice, a panel of monoclonal antibodies was used to identify the following subsets: cn4+44+, cns+44+ and cns+11b+.11. 18 Spleens from TBM contained essentially the same percentage of T cells of both the CD4+CD44+ and the CD8+CD44+ phenotypes as that of naive mice (fig. 6). Little or no change was observed in the CDS+ cells in any of the treatment groups. The percentage of CD4+44- cells was lower in TBM than in naive mice but was normalized to some degree by all four treatments. The CD4+44+ cell type was also elevated in the four treatment groups relative to naive or untreated TBM.
Cells froEl tumor uuuLuus s:111n,Pr,1P, inguinal LNs from these same mice were also v~.,uu.,wcv., and quite similar patterns of change in the CD44+ subsets were observed (data not shown). Cells coexpressing CD8 and CDllb are also thought to be memory T cells in the murine system. This cell population was examined; however, there were no changes observed for either spleen or node (data not shown). The above analyses were performed on lymphocytes only (other cells were gated out). Alternatively, the combination treatment might augment nonspecific host response. Therefore, the total CDllb+ population, composed primarily of cells from myeloid/macrophage lineage, 19 was also evaluated. As shown in figure 7, the levels of CDllb+ cells rose in the spleen but not LN following TR+TV and, to a greater extent, following CY +TR or CY +TR+TV. DISCUSSION
The perioperative period is optimal for anticancer chemoimmunotherapy because, at this critical stage: 1) the tumor burden is at its minimum; 2) the ongoing tumor-induced immunosuppression of the host has been temporarily terminated by tumor resection; 3) there may have been a certain degree of host specific antitumor immunity already elicited prior to tumor resection. Therefore, theoretically, treatment, either singly or in combination, that can eliminate the residual tumorinduced immunosuppression and/or augment the host specific antitumor immunity may achieve therapeutic benefit. In this study, the major purpose of using low dose CY pretreatment was to take advantage of the immunopotentiating effect derived from its selective toxicity to precursors of suppressor T cells. 6 • 7 \Ve were able to demonstrate in the studies assessing cytotoxicity of in vitro stimulated effectors that CY pretreatment at the dose of 100 mg./kg., intraperitoneally, on day 15 can significantly enhance the cytolytic activity of splen ocytes against MBT-2 cells. This effect of CY, as assessed in vitro, appears to be somewhat transient since augmentation of cytotoxicity was seen 2 but not 16 days after CY injection. In studies assessing in vivo tumor growth following tumor reimplantation, CY treatment in combination with TR and TV was found to be beneficial. Evidence from the phenotypic analysis shows that CY pretreatment not only abolishes the downregulating effect of tumor on memory T cells in the spleen, but also induces an increase in phagocytes (CDllb+ cells). These latter cells may suppress the tumor growth through nonspecific mechanisms. Postoperative immunization with autologous tumor vaccine is believed to further stimulate memory T cells and CTLs by providing an additional source of tumor associated antigen which, in turn, enhances the host specific antitumor immunity. The fact that naive C3H/He mice immunized with irradiated MBT-2 cells are protected against viable MBT-2 cells has been 20 but under conditions examined in Iviorales et our study tumor implantation) TV alone did not significantly alter tumor incidenceo The MBT-2 tumor line is known to be a highly malignant and poorly differentiated murine transitional cell carcinoma. 12 Sixty Gy of irradiation is lethal for MBT--2 cells based on the lack of growth in culture or in vivo. Nevertheless, evidence from the in vitro cytotoxicity assays showed that, during the initial 5 days after irradiation, the cells remain intact. Furthermore, these cells appear to be metabolically active, in so far as they were shown to continue to secrete soluble suppressive factors during that period (fig. 3). Using these factor-secreting irradiated tumor cells as the source of tumor vaccine could have contributed to tumorinduced immunosuppression, especially when TV was administered at a critical stage during the generation of immunocompetent cells. Consistent with this, in this study, TV alone following tumor implantation did not have a significant effect on tumor incidence at 60 days. Subsequent phenotypic analysis indicated that postoperative TV immunization, however, in-
duced more CD44 + T cells is, memory and cells having the CDllb+ phenotype (macrophages, granulocytes, or NK cells). Under these conditions, the stimulation of specific and nonspecific immunity by TV apparently overbalanced the effect of soluble suppressor factors. One approach to avoid suppression by soluble factors would be to prepare a vaccine using cell membrane fragments instead of whole cells. This would be worth trying in this system since it was reported previously 19 that membrane preparations of MBT-2 cells effectively protect mice against challenge with viable MBT-2. We therefore concluded that (1) under certain circumstances, preoperative CY treatment can enhance the host specific antitumor activity and temporarily provide an optimal environment for immunomodulation; (2) immunization with irradiated autologous tumor cells is not always beneficial; (3) the combination of the two can be effective under the condition of minimal residual tumor burden after surgery (::S10 6 tumor cells in this model). It may be particularly useful to determine whether the cells in a TV are metabolically inactive, because if they are not, they may contribute untoward effects. Acknowledgements. The authors would like to express their sincere thanks to Cheryl Eppolito for her technical assistance and to Jane Meer for her assistance in data management. The review and comments of Dr. William Greco are gratefully acknowledged. REFERENCES
1. Salisbury, A. J., Kinna, J. A., Griffiths, J. D. and Naunton-Morgan,
2. 30 4. 5. 6.
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C.: Circulating cancer cells during excising of carcinomas of the rectum and colon with high ligation of the inferior mesenteric vein. Surg. Gynaecol. Obst., 120: 1266, 1965. Glaves, D., Huben, R. P. and Weiss, L.: Haematogenous dissemination of cells from human renal adenocarcinomas. Br. J. Cancer, 57: 32, 1988. Maccubbin, D. L., Mace, K. F., Ehrke, M. J. and Mihich, E.: Modification of host antitumor defense mechanisms in mice by progressively growing tumor. Cancer Res., 49: 4216, 1989. Pollock, R. E. and Roth, J. A.: Cancer-induced immunosuppression: implication for therapy? Semin. Surg. Oncol., 5: 414, 1989. Salo, M.: Effects of anaesthesia and surgery on the immune response. Acta Anaesthesiol. Scand., 36: 201, 1992. Awwad, M. and North, R. J.: Cyclophosphamide-induced immunologically mediated regression of a cyc!ophosphamide-resistant murine tumor: a consequence of eliminating precursor L3T4+ suppressor T-cells. Cancer Res., 49: 1649, 1989. Ozer, H., Cowens, J. W., Colvin, M., Nussbaum-Blumensen, A. and Sheedy, D.: In vitro effect of 4-hydroperoxycyclophosphamide on human immunoregulatory T subset function. J. Exp. Med., 155: 276, 1982. Hanna, M. Go, Jr. and Peter, L. C.: Specific immunotherapy of established visceral micrometastases by BCG-tumor cell vaccine alone or as an adjunct to surgeryo Cancer, 42: 2613, 1978. Schirrmacher, V. and Heicappel, R.: Prevention of metastatic spread by postoperative immunotherapy with virally modified autologous tumor cells. IL Establishment of specific systemic antitumor immunity. Clin. Exp. Metastasis, 5: 147, 1987. Hoover, H. C., Surdyke, M. Go, Dangel, R. B., Peter, L. C. and Hanna, M. G., Jr.: Prospectively randomized trial of adjuvant active-specific immunotherapy for human colorectal cancer. Cancer, 55: 1236, 1985. Berd, D., Maguire, H. C., Jr., McCue, P. and Mastrangelo, M. J.: Treatment of metastatic melanoma with autologous tumor-cell vaccine: clinical and immunologic results in 64 patients. J. Clin. Oncol., 8: 1828, 1990. Mickey, D. D., Mickey, G. H., Murphy, W. M., Niel!, H. B. and Soloway, M. S.: In vitro characterization of four N-[4-(5-nitro2-furyl)-2-thiazolyl] formamide (FANFT) induced mouse bladder tumors. J. Urol., 127: 1233, 1982. Tomazic, V., Ehrke, M. J. and Mihich, E.: Augmentation of the development of immune responses of mice against allogeneic tumor cells after adriamycin treatment. Cancer Res., 41: 3370, 1981. Berleth, E., Ehrke, M. J., Dolnick, B. and Mihich, E.: Quantitation of cytokine mRNA levels in tissues from in vivo sources using
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PERIOPERATIVE CHEMOIMMUNOTHERAPY IN MURINE BLADDER CANCER
reverse transcription (RT)/polymerase chain reaction (PCR) assays. Eur. J. Cancer, 29A: S47, 1993. 15. Dolnick, B. J., Zhang, Z. G., Hines, J. D. and Rustum, Y. M.: Quantitation of dihydrofolate reductase and thymidylate synthase mRNAs in vivo and in vitro by polymerase chain reaction. Oncol. Res., 4: 65, 1992. 16. Ito, N., Kawata, S., Tamura, S., Takaishi, K., Shirai, Y., Kiso, S., Yabuuchi, I., Matsuda, Y., Nishioka, M. and Taru, S.: Elevated levels of transforming growth factor fJ messenger RNA and its polypeptide in human hepatocellular carcinoma. Cancer Res., 51: 4080, 1991. 17. Budd, R. C., Cerrottini, J. C., Horvath, C., Bron, C., Pedrazzini,
T., Howe, R. C. and MacDonald, R.: Distinction of virgin and memory T lymphocytes: stable acquisition of the Pgp-1 glycoprotein concommitant with antigenic stimulation. J. lmmunol., 138: 3120, 1987. 18. McFarland, H. I., Nahill, S. R., Maciaszek, J. W. and Welsh, R. M.: CDllb(Mac-1): a marker for cos+ cytotoxic T cell activation and memory in virus infection. J. Immunol., 149: 1326, 1992. 19. Yurochko, A. D., Pyle, H. R. and Elgert, K. D.: Changes in macrophage populations: phenotypic differences between normal and tumor-bearing host macrophages. Immunobiol., 178: 416, 1989. 20. Morales, A., Djeu, J. and Herberman, R. B.: Immunization by irradiated whole cells or cell extracts against an experimental bladder tumor. Invest. Urol., 17: 310, 1980.
Vol. 151, 1680-1686, June 1994 Printed in U.S. A.
EFFECT OF PERIOPERATIVE CHEMOIMMUNOTHERAPY WITH CYCLOPHOSPHAMIDE AND AUTOLOGOUS TUMOR VACCINE IN MURINE MBT-2 BLADDER CANCER TZONG-SHIN TZAI, ROBERT P. RUBEN, GINTARAS ZALESKIS, ERICA S. BERLETH, M. JANE EHRKE* AND ENRICO MIRICH From the Department of Urologic Oncology and Grace Cancer, Drug Center, Roswell Park Cancer Institute, Buffalo, New York
ABSTRACT
The in vitro cytotoxic activity of splenocytes from C3H/He mice implanted subcutaneously with 106 syngeneic MBT-2 tumor cells on day O was significantly enhanced after cyclophosphamide (100 mg./kg., intraperitoneally) given 2 days before tumor resection on day 17, with or without active specific immunization with BCG plus autologous irradiated tumor cells (vaccine) 1 week after tumor resection. Furthermore, a significantly lower tumor incidence was seen in mice challenged with 105, but not 106 , tumor cells per mouse 24 hours after tumor resection on day 17 and treated with cyclophosphamide on day 15 and postoperatively with vaccine than was found in nontreated tumor resected mice. Phenotypic analysis of cells from spleen showed that cyclophosphamide pretreatment and postoperative vaccine, either singly or in combination, induced a significant increase of both CD44+ memory T cells and CDllb+ myeloid/macrophage cells. Thus, in addition to a specific antitumor immune response, a nonspecific cytolytic mechanism may also play a role in the observed antitumor effect. KEY WORDS: drug therapy, immunotherapy, cyclophosphamide
Treatment of moderate to advanced stages of cancer by surgery alone usually fails because of local recurrence and/or distant metastasis. The major reasons for the failure are believed to be: (1) tumor cell dissemination into the circulation during surgical manipulation;1· 2 (2) undetected residual local tumor or systemic micrometastasis of tumor; (3) further deterioration of the already suppressed host antitumor immune defenses due to surgical and/or anesthetic stress. 3- 5 Theoretically, immune stimulation immediately before or after tumor resection should reduce the chance for any residual tumor cells to grow. Cyclophosphamide (CY), when administered at an appropriately low dose, has been shown to have immunoaugmenting effects by being relatively toxic to the precursors of suppressor T cells, while sparing cytotoxic T lymphocyte precursor cells. 6 • 7 Active specific immunization, using a vaccine prepared by irradiated autologous tumor cells mixed with Bacillus CalmetteGuerin (BCG), has been demonstrated to induce specific antitumor responses in tumor-bearing animals 8 • 9 and to improve both disease-free interval and survival of patients with colorectal cancer and melanoma. 10• 11 In this study the C3H/He-MBT2 syngeneic murine bladder tumor model was used. The situation of bladder cancer patients treated with surgery was mimicked, and the perioperative immunomodulating effects of CY pretreatment, with or without postoperative tumor vaccine (TV) immunization, were studied. MATERIALS AND METHODS
Animals. Female C3H/HeJCr mice, 4 weeks old, were obtained from the Mammalian Genetics and Animal Production Section, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland. Mice received free access to mouse chow and water and were studied between 6 and 8 weeks of age. Tumor cell lines. The MBT-2 murine transitional cell carciAccepted for publication December 10, 1993. * Requests for reprints: Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263. This work was supported in part by grants CA15142 and CA16056 from the National Cancer Institute.
noma was originally induced by N-(4-(Nitro-2-furyl)-2-thiazolyl) formamide (FANFT) administration to C3H/He mice. 12 Cells from this tumor line were maintained in RPMI 1640 plus 10% fetal calf serum (FCS). Media and reagents. RPMI 1640 supplemented with 10% heat inactivated FCS (Hyclone Laboratories, Logan, Utah), 1 mM. sodium pyruvate, 0.1 mM. nonessential amino acids, 25 mM. Hepes, gentamicin 50 ,ug./ml., 2.0 mM. glutamine and 50 ,uM. 2-mercaptoethanol is termed complete medium. The endotoxin levels in the FCS were in the range of 0.005 to 0.033 ng./ ml. Cyclophosphamide (Sigma Chemical Co., St. Louis, Missouri, Lot No. 19F02541) was dissolved in 0.9% saline to a concentration of 10 mg./ml. Recombinant human interleukin2 (IL-2) was a gift from E.I. DuPont de Nemours Co., Inc., Glenolden, Pennsylvania (Lot No. 0024C). Based on published information and as confirmed in this laboratory by a standard bioassay, 1 DuPont unit equals 30 BRMP units, and the specific activity of this recombinant material is 3700 BRMP units per ,ug. protein. Bacillus Calmette-Guerin (BCG) (Tokyo strain, Japan BCG Co., Tokyo, Japan) was suspended in 0.9% saline at a concentration of 10 mg./ml. The BCG (1 mg. in 0.1 ml. saline) was mixed with 106 irradiated (60 Gy) MBT-2 cells (IRMBT-2) as the TV and was injected subcutaneously into the abdominal wall of the mice. MBT-2 cells which had received 60 Gy x-irradiation failed to develop tumors in naive mice and did not proliferate in culture. Therefore, this is considered a lethal dose. Tumor cell implantation. MBT-2 tumor cells, harvested from culture flasks by trypsinization (Trypsin 0.25%, Gibco, Grand Island, New York), were washed two times in complete medium, counted (Trypan blue exclusion) and diluted to 10 7/ml. in phosphate buffered saline (PBS). A 0.1 ml. aliquot was implanted subcutaneously into the lower abdominal area of each mouse. Sublethal whole body x-irradiation. A Siemens 250 KV X-ray unit was used. Mice were placed into a cylindrical Plexiglas restrainer in the axis of the X-ray beam. The skin-to-source distance was 47.5 cm., and the dose rate was 1.15 Gy per minute in air. The x-irradiated mice received a single nonlethal dose of 4 Gy on the day of tumor implantation.
1680
1681
PER!OPERATIVE CHEMOYMMUNOTHERAPY IN MURINE BLADDER CANCER
~
Tumor inoculatioo
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A1110 = = = = = = = = = = I = = = = = = = = = = = = = = = = = = I N , A2 I 10 = = = = = = = = = = I = = = = = V = = = = = = = = = = = = I N , A3111 , T = = = = = = R = I IN, A4 I 11 , T = = = = = = = R = I
AS I 9 'T = C Y = R = I A619 , T = C Y = R = I
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B1110 = = = = = = = = = = I = = = = = = = = = = = = = = = = = = IN, B2110 = = = = = = = = = = I = = = = = V = = = = = = = = = = = = IN,
B3111, T=======R= I B4! 11 , T =======R = I B519 , T = C Y = R = I B6! 9 , T = C Y = R = I
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FIG. 1. Schemes of treatment protocols of in vivo tumor challenge studies. T: 106 tumor cells subcutaneous!¥ inoculated on day O; CY: Cyclophosphamide, 100 mg./kg., intraperitoneally on day 15; R: Resection of tumor on day 17; I: Implantation of 10 (Gr.A) or 105 (Gr.Bl viable MBT-2 tumor cells subcutaneously on day 18; V: Tumor vaccine subcutaneous injection on day 24; CTL: Cytotoxic T-lymphocyte assay; PH: Phenotyping for immune memory cells: co4+44+, co3+44+ and CD8+nb+. IN: Tumor incidence 60 days after reimplantation on day 18. Individual panels describe experimental design and number of mice per group for experiments yielding data shown as follows: Panel A for figs. 2 and 3; Panel B for figs. 4, 6, and 7; Panel C for fig. 5 and table 2.
TABLE
1. Effect of a sublethal dose (4 Gy) whole body irradiation on
No. of Tumor Cells Inoculated 10 105 10• 103
102 10 1
8/12 3/12 2/12 3/12 3/12 3/12
= 72) (66.7%) (25.0%) (16.7%) (25.0%) (25.0%) (25.0%)
Irradiated (n = 72) 12/12 12/12 10/12 7/12 8/12 8/12
(100.0%) (100.0%) (83.3%) (58.3%) (66.7%) (66.7%)
CY(+) IRMBT-2(-) " CY(-) IRMBT-2(+) ,w CY(+) IRMBT-2( +)
**
@
35
P" value (n
6
MBT-2
o CY(-) IRMBT-2(-)
Percent Tumor Incidence by Day 45 Nonirradiated
Target
40
the tumor incidence
*
Q) [/]
0.0466 0.0002 0.0017 0.1069 0.0498 0.0498
"Fisher's exact test; p value: irradiated versus nonirradiated.
/Cl
30
Q.) Q.)
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**
p< 0.01
*
p< 0.05
25 20
()
:;::
Tumorigenicity and immunogenicity study of MBT-2 in C3H/ HeJCr mice. One hundred and forty-four mice were randomly allocated to two groups. One group received 4 Gy of sublethal whole body x-irradiation immediately before tumor cell implantation. The two groups (x-irradiated and nonirradiated) were then divided into 6 subgroups (12 mice per subgroup) which were implanted with 106 , 105, 104 , 103 , 10 2 , 101 MBT-2 cells per mouse. Tumor growth was monitored twice a week. Surgical tumor resection and treatment with CY with or without TV. Mice implanted with 106 MBT-2 tumor cells on day O were divided into 5 groups: one group [tumor bearing mice (TBM)] received no further treatment; 2 groups received CY (100 mg./kg. intraperitoneally) treatment on day 15 and 2 groups received tumor vaccine on day 24. All groups except the TBM group underwent tumor resection on day 17. Ketamine
() Q.)
15
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~
10 5
12/1
25/1
50/1
100/1
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FIG. 2. Effect of CY pretreatment and/or IRMBT-2 on specific cytotoxicity of splenocytes from day 17 TBM. Splenocytes (5 x 106/ ml.) obtained on day 17 from CY (100 mg./kg., intraperitoneally, day 15) treated or nontreated TBM (experimental design shown in fig. 1, A) were cocultured in presence of IL-2 (0.5 U/ml.) with/without IRMBT-2 (R/S ratio: 100/1) for 5 days. Assayed in 4 hour 51 Cr release assay. Data obtained at four E:T ratios shown.
1682
PERIOPERATIVE CHEMOIMMUNOTHERAPY IN MURINE BLADDER CANCER 50
TABLE
E/T 100/1 Target: MBT-2 Effector: Splenocytes
(l)
~
Gr'
40
2. In vivo tumor challenge study
Treatmenth
* P< 0.05
(l)
.;
** P< 0.01
0::
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20
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(l)
0. rt)
Al A2 A3 A4 A5 A6
Control' TV" TR TR+TV CY+TR CY+TR+ TV
Bl B2 B3 B4 B5 B6
Control' TV' TR TR+TV CY+TR CY+TR+TV
~ 10
D2-S
D3-S
D4-S
FIG. 3. Suppression of specific cytolytic activity of splenocytes by fresh or cultured irradiated MBT-2 tumor cells and their conditioned medium. Splenocytes (5 X 106/ml.) pooled from 3 to 4 day-17 TBM (experimental design was as shown on first line of fig. 1, A) were cultured in all cases with 0.5 U IL-2/ml. for 5 days plus: (1) complete medium alone (control); (2) fresh IRMBT-2 at R:S ratio of 100/1 (IRMBT-2); (3) cellular component recovered after IRMBT-2 (5 X 103 cells/0.2 ml./well) were cultured for 5 days with daily change of medium (D5-C); supernatant harvested on days 1, 2, 3 and 4 from wells of cultured IRMBT-2 (Dl-S, D2-S, D3-S, D4-S); final concentrations in wells were 25% or 50% of total volume.
LN
Spleen (l)
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co
70
E/T
OJ 60
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Target , MBT- 2
60
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50
40
40
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8/10 9/10 2/11 2/11 1/9 1/9
(80.0%) (90.0%) (18.2%) (18.2%) (11.1 %) (11.1%)
0.5 0.0073 0.0073 0.0045 0.0045
(50.0%) (10.0%) (36.4%) (9.1%) (11.1 %) (0.0%)
0.0704 0.4250 0.055 0.0913 0.0217
105 cells
Dl-S
(l)
p• value (vs. control)
106 cells
'"" lJ'.)
S
Tumor Incidence 60 days after MBT-2 reimplantation on Day 18'
Q' f-
i"
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>-
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+
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FIG. 4. In vitro cytotoxicity assay of effector cells from spleen and LNs. Lymphocytes obtained on day 31 from spleen and bilateral superficial inguinal LN were cultured in presence of IL-2 (0.5 U/ml.) for 5 days. Five experimental groups (experimental design and number of mice per group shown in fig. 1, B) were (1) day-31 TBM; (2) TR: tumor resected on day 17; (3) TR+TV: TR followed 1 week later by tumor vaccine (TV) immunization; (4) CY+TR: CY 100 mg./kg. intraperitoneally given 2 days before TR; (5) CY +TR+TV: mice received all three treatments. This experiment has been repeated 4 times with similar results.
(2.5 mg./kg.) intraperitoneally was used as the general anesthetic during surgery. On day 31 the mice in the 5 groups and age-matched naive mice were sacrificed to assess the effector functions of spleen and lymph node cells ex vivo. Preparation of single cell suspensions from spleen and lymph nodes. Spleen and bilateral superficial inguinal lymph nodes (LN) were removed aseptically on the day of sacrifice. Single cell suspensions were prepared as described previously. 13 Generation and assay of cytolytic effector cells. Aliquots (0.1 ml.) of spleen and lymph node cells (5 x 106 /ml. complete medium) were added to six wells of a 96-well round bottom culture plate. Aliquots (0.1 ml.) of rIL-2 (1.0 U/ml.) were added to each well giving a final concentration of0.5 U/ml. The plates were incubated for 5 days. The cultured cells were then serially
5/10 1/10 4/11 1/11 1/9 0/9
'The experimental design and numbers of mice per group are shown in figure lC. Mice in groups A3-A6 and B3-B6 received 106 MBT-2 tumor cells, subcutaneously, on day 0. h CY: cyclophosphamide (100 mg.jkg. intraperitoneally on day 15). TR: Tumor resection on day 17. TV: tumor vaccine (BCG + 106 IRMBT-2) immunization on Day 24. ' All mice in the two sets of 6 subgroups received subcutaneous inoculation with viable MBT-2 tumor cells [10 6 cells (Gr.A) or 105 cells (Gr.BJ, respectively] on day 18 (24 hours after tumor resection). • Fisher's exact test. • Naive mice (did not receive tumor on day 0).
diluted (2-fold) into wells containing complete medium only, and the cytolytic activity was assessed in a standard 4 hour 51 Cr-release assay described elsewhere. 3 • 13 In certain experiments, in addition to the IL-2, IRMBT-2 stimulator cells (S) were added to the spleen and LN responder cells (R) at a final R:S ratio of 100 to 1. Quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) assay for transforming growth factor-beta (TGF-(3). The assay involves a competitive template technique for quantitation and has been recently described. 14 It is based on the technique reported by Dolnick et al. 15 Briefly, a known amount of internal standard, which competes identically for all reactants, is added to cellular RNA and subjected to RT/PCR. The standard is engineered to contain a unique restriction enzyme site. Therefore, following amplification and digestion, it is possible to determine the amount of the product derived from cellular versus standard RNA. Phenotyping of the splenocytes and LN cells: a three-color analysis. Aliquots of cells were stained for simultaneous threecolor analysis on a FACScan flow cytometer (Becton-Dickinson, San Jose, California). The following monoclonal antibodies were used: FITC labeled anti-CDllb (Mac-1), PE conjugated anti-CD4 and anti-CDS, biotinylated anti-CDS (all purchased from Caltag, San Francisco, California) and FITC labeled antiCD44 (Pharmingen, San Diego, California). Streptavidin-Red 670 (Gibco) was added to visualize biotinylated anti-CDS as a second step. Ten thousand events were acquired in list mode. Lysys II and Paint-A-Gate software (Becton-Dickinson) were used for subset evaluation. In vivo tumor challenge study. The experimental design is outlined schematically in figure 1, C. Eight mice with palpable tumors, which were induced by subcutaneous implantation of 106 viable MBT-2 tumor cells on day 0, had their tumors resected on day 17. Thirty-six of them had been pretreated with CY (100 mg./kg., intraperitoneally) two days (day 15) before tumor resection. For comparative purposes, these 36 CY treated and the 44 untreated mice, together with 40 naive mice, were randomized into parallel subgroups as shown in the scheme in figure 1, C. All mice were challenged by subcutaneous implantation of either 106 (six "A" groups) or 105 (six "B" groups) viable tumor cells per mouse on day 18 (24 hours after
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tumor resection) at a site adjacent to the surgical wound. Six days later (day 24) one-half of the mice received a subcutaneous injection of TV as indicated. Tumor outgrowth and survival of mice were monitored daily. Statistical analysis. Independent Student's t test and Fisher's Exact test using SPSSPC+ software were used for data analysis. RESULTS
Tumorigenicity and immunogenicity of MBT-2 tumor cells. Initial studies had indicated that, following subcutaneous injection of 10 6 MBT-2 cells, only 60 to 70% of C3H/HeJCr mice developed progressive disease. This low tumor incidence suggested that host antitumor defenses might be involved. An experiment was carried out, therefore, comparing tumor incidence in nonirradiated mice to that in immunosuppressed, lowdose, whole body irradiated mice to determine the possible contribution of host antitumor defense to the low tumor incidence seen. The number of tumor cells inoculated was varied
logarithmically from 10 1 to 106 , and the percentage of tumor incidence observed by day 45 was calculated for both the whole body irradiated (4 Gy) and nonirradiated groups. It is clear from table 1 that a) for both groups, the percent tumor incidence increased in a dose-responsive manner with inoculum size and that b) the dose-response curve for the irradiated group lies to the right of that for the nonirradiated group. Thus, the immunologically deprived animals were clearly more susceptible to the challenge of inoculated tumor cells throughout the range of inocula tested. Separate tests of statistical significance (Fisher's Exact Test) comparing the nonirradiated versus irradiated groups for each inoculum size confirmed the overall trend. Only one separate analysis, that for 103 inoculated cells, missed being significant at the 0.05 level. Since low-dose, whole body x-ray irradiation is known to temporarily ablate the host's specific antitumor immunity, but to have much less effect on macrophage/monocyte mediated defenses, the results obtained suggest that the MBT-2 tumor is immunogenic in the syngeneic C3H/HeJCr host.
1684
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The x-irradiated MBT-2 tumor cells remain metabolically active and immunosuppressive for at least 5 days. One approach used to demonstrate that a host is mounting a specific response to a syngeneic tumor is to challenge lymphocytes from TBM in culture with metabolically inactivated (x-irradiated) tumor cells and assess the ability of these lymphocytes to lyse syngeneic tumor target cells. The addition of fresh x-irradiated (60 Gy) MBT-2 cells (IRMBT-2), to in vitro response cultures with splenocytes from untreated day-17 TBM (fig. 2), however, was found to significantly reduce the level of cytolytic activity that developed. The administration of a dose of CY, which has been reported6 to be immunoaugmenting, significantly elevated the level of lytic activity attained with or without the addition of x-irradiated tumor. In each case, however, the levels attained in the presence of x-irradiated tumor were lower than those in the corresponding culture without its addition, suggesting that the tumor was immunosuppressive. Since this finding appeared to be in disagreement with the in vivo tumor incidence findings, further experiments were carried out. In these experiments it was found that the cellular component of day-5 cultured IRMBT-2, or supernatants harvested daily from the same cultured IRMBT-2 cells, when added at the beginning of in vitro response cultures with splenocytes from day-17 TBM, all suppressed the generation of specific cytotoxic activity (fig. 3). In a separate experiment, it was found that 24 hour supernatants from nonirradiated MBT-2 cells were also suppressive but markedly less so than those from x-irradiated cells (data not shown). Based on the above findings, the considerable literature indicating a role for (TGF-/n in tumor-induced suppression, and the fact that elevated mRNA expression and protein production have been shown to be associated, 16 quantitative RT/ PCR for TGF-/3 mRNA was carried out with RNA extracted from nonirradiated and x-irradiated MBT-2 cells. No substantial difference was seen in the level of message between the two conditions. Studies to identify the soluble factor are continuing. Effect of CY pretreatment with or without postoperative TV immunization on the antitumor activity of splenocytes and LN cells from TBM. An alternative approach to demonstrate specific host responses is to examine the ability of cells from TBM undergoing curative treatments to lyse syngeneic tumor target cells. As shown in fig. 4, the specific cytolytic activity of effector cells generated from both spleen and tumor draining LN obtained from TBM sacrificed on day 31 was increased significantly (p <0.001 and p <0.01, respectively) when compared with that of cells from control (naive, nontumor bearing) mice.
They both decreased markedly (p <0.001, p <0.01) in the group which had undergone tumor resection (TR) two weeks (day 17) earlier. Relative to TR alone, the preoperative (day 15) administration of CY followed by postoperative (day 24) TV immunization significantly enhanced the antitumor activity of effector cells in spleen (p <0.001) but not in LN (p >0.05). Similarly, relative to the TR alone, the adjuvant postoperative immunization with TV also induced a significant augmentation of the antitumor activity of splenocytes (p <0.001) but not of LN cells (p >0.05). In contrast, when the effect of preoperative CY treatment was assayed at this point (day 31), 2 weeks after tumor resection and 16 days after CY injection, there was no difference in spleen (p >0.05) or LN (p >0.5) cells relative to those from the TR alone groups. However, when assessed 2 (day-17 TBM, fig. 2) or 6 days (day-21 TBM, data not shown) after CY treatment, augmentation was seen. In vivo tumor challenge study. In the animal model under study surgical resection of the tumor was complete in the large majority of cases. Since this is not always the case clinically, a technique was chosen to mimic the situation of possible residual tumor due to incomplete surgical removal of a primary bladder cancer or the presence of micrometastases at the time of surgery. The effect of the perioperative treatments on host specific antitumor immunity was examined by subcutaneous reimplantation of either 106 or 105 tumor cells per mouse 24 hours after surgical removal of the primary tumor as outlined in figure l,C. It was found 60 days later that tumor incidence in the control groups was greater following implantation with 106 cells (Al group) than with 105 cells (Bl group, 5 of 10 or 50% versus 8 of 10 or 80%). This means that the control group to which all treatment groups are compared is smaller for those receiving 105 cells than for those receiving 106 cells. For the mice receiving 106 cells the four (A3-6) treatment groups (day-17 TR, TR+TV, CY +TR and CY +TR+TV) all demonstrated augmented (p <0.01, versus control) antitumor activity (table 2). There was no statistical difference, however, between the four groups in terms of tumor incidence or survival times (table 2 and fig. 5). In groups of mice challenged with 105 tumor cells per mouse, TV immunization caused suppression of tumor growth in both naive mice [from 50% (Bl) to 10% (B2)] and tumor-resected mice [from 36.4% (B3) to 9.1% (B4)]. However, the differences were not statistically significant [p >0.05 (Bl versus B2), p >0.1 (B3 versus B4)]. The lack of clear statistical significance for treatments of groups B3-B5 was due to the low incidence of tumor in naive mice when implanted with 105 MBT-2 tumor cells per mouse, since the outcome of treatment in these groups did not differ from that seen in groups A3-A5. Nevertheless, the triple treatment (group B6) was significantly better (p <0.025) than control, and the fact that not a single mouse in group B6 had tumor growth indicates the therapeutic efficacy of combined therapy of CY pretreatment and postoperative TV immunization with minimal residual tumor burden (10 5 cells) after surgery. Phenotypic analysis of leucocyte subsets. If specific host defense mechanisms were involved in the overall efficacy of the combination treatments, then one would expect development of memory T cells. To determine if the effect of CY pretreatment with or without postoperative TV immunization resulted in phenotypically definable memory subsets in spleen and/or LN of treated mice, a panel of monoclonal antibodies was used to identify the following subsets: cn4+44+, cns+44+ and cns+11b+.11. 18 Spleens from TBM contained essentially the same percentage of T cells of both the CD4+CD44+ and the CD8+CD44+ phenotypes as that of naive mice (fig. 6). Little or no change was observed in the CDS+ cells in any of the treatment groups. The percentage of CD4+44- cells was lower in TBM than in naive mice but was normalized to some degree by all four treatments. The CD4+44+ cell type was also elevated in the four treatment groups relative to naive or untreated TBM.
Cells froEl tumor uuuLuus s:111n,Pr,1P, inguinal LNs from these same mice were also v~.,uu.,wcv., and quite similar patterns of change in the CD44+ subsets were observed (data not shown). Cells coexpressing CD8 and CDllb are also thought to be memory T cells in the murine system. This cell population was examined; however, there were no changes observed for either spleen or node (data not shown). The above analyses were performed on lymphocytes only (other cells were gated out). Alternatively, the combination treatment might augment nonspecific host response. Therefore, the total CDllb+ population, composed primarily of cells from myeloid/macrophage lineage, 19 was also evaluated. As shown in figure 7, the levels of CDllb+ cells rose in the spleen but not LN following TR+TV and, to a greater extent, following CY +TR or CY +TR+TV. DISCUSSION
The perioperative period is optimal for anticancer chemoimmunotherapy because, at this critical stage: 1) the tumor burden is at its minimum; 2) the ongoing tumor-induced immunosuppression of the host has been temporarily terminated by tumor resection; 3) there may have been a certain degree of host specific antitumor immunity already elicited prior to tumor resection. Therefore, theoretically, treatment, either singly or in combination, that can eliminate the residual tumorinduced immunosuppression and/or augment the host specific antitumor immunity may achieve therapeutic benefit. In this study, the major purpose of using low dose CY pretreatment was to take advantage of the immunopotentiating effect derived from its selective toxicity to precursors of suppressor T cells. 6 • 7 \Ve were able to demonstrate in the studies assessing cytotoxicity of in vitro stimulated effectors that CY pretreatment at the dose of 100 mg./kg., intraperitoneally, on day 15 can significantly enhance the cytolytic activity of splen ocytes against MBT-2 cells. This effect of CY, as assessed in vitro, appears to be somewhat transient since augmentation of cytotoxicity was seen 2 but not 16 days after CY injection. In studies assessing in vivo tumor growth following tumor reimplantation, CY treatment in combination with TR and TV was found to be beneficial. Evidence from the phenotypic analysis shows that CY pretreatment not only abolishes the downregulating effect of tumor on memory T cells in the spleen, but also induces an increase in phagocytes (CDllb+ cells). These latter cells may suppress the tumor growth through nonspecific mechanisms. Postoperative immunization with autologous tumor vaccine is believed to further stimulate memory T cells and CTLs by providing an additional source of tumor associated antigen which, in turn, enhances the host specific antitumor immunity. The fact that naive C3H/He mice immunized with irradiated MBT-2 cells are protected against viable MBT-2 cells has been 20 but under conditions examined in Iviorales et our study tumor implantation) TV alone did not significantly alter tumor incidenceo The MBT-2 tumor line is known to be a highly malignant and poorly differentiated murine transitional cell carcinoma. 12 Sixty Gy of irradiation is lethal for MBT--2 cells based on the lack of growth in culture or in vivo. Nevertheless, evidence from the in vitro cytotoxicity assays showed that, during the initial 5 days after irradiation, the cells remain intact. Furthermore, these cells appear to be metabolically active, in so far as they were shown to continue to secrete soluble suppressive factors during that period (fig. 3). Using these factor-secreting irradiated tumor cells as the source of tumor vaccine could have contributed to tumorinduced immunosuppression, especially when TV was administered at a critical stage during the generation of immunocompetent cells. Consistent with this, in this study, TV alone following tumor implantation did not have a significant effect on tumor incidence at 60 days. Subsequent phenotypic analysis indicated that postoperative TV immunization, however, in-
duced more CD44 + T cells is, memory and cells having the CDllb+ phenotype (macrophages, granulocytes, or NK cells). Under these conditions, the stimulation of specific and nonspecific immunity by TV apparently overbalanced the effect of soluble suppressor factors. One approach to avoid suppression by soluble factors would be to prepare a vaccine using cell membrane fragments instead of whole cells. This would be worth trying in this system since it was reported previously 19 that membrane preparations of MBT-2 cells effectively protect mice against challenge with viable MBT-2. We therefore concluded that (1) under certain circumstances, preoperative CY treatment can enhance the host specific antitumor activity and temporarily provide an optimal environment for immunomodulation; (2) immunization with irradiated autologous tumor cells is not always beneficial; (3) the combination of the two can be effective under the condition of minimal residual tumor burden after surgery (::S10 6 tumor cells in this model). It may be particularly useful to determine whether the cells in a TV are metabolically inactive, because if they are not, they may contribute untoward effects. Acknowledgements. The authors would like to express their sincere thanks to Cheryl Eppolito for her technical assistance and to Jane Meer for her assistance in data management. The review and comments of Dr. William Greco are gratefully acknowledged. REFERENCES
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PERIOPERATIVE CHEMOIMMUNOTHERAPY IN MURINE BLADDER CANCER
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