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*
Biologic Therapeutics Program, University of Pittsburgh Cancer Institute, and
Thomas E. Starzl Transplantation Institute, Pittsburgh, PA 15213; and
Immunex R&D Corp., Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 promotes the activation and generation of CTL, stimulates NK activity and macrophage function, and regulates Th1 cell generation (8, 9, 10). The antitumor activities of IL-12 have been studied extensively in a number of animal models with promising results (11), but its clinical application was made difficult by the occurrence of unusual systemic toxicity observed at high doses of the cytokine (12, 13). Thus, alternative strategies of IL-12 application will be important for the future development of IL-12-based immunotherapies. Moreover, IL-12 has been shown to have a synergistic interaction with FL in stimulating the growth and expansion of early murine bone marrow progenitor cells in vitro (14), suggesting the possible use of combination of these cytokines in a tumor immunotherapy.
We have examined the effect of FL treatment on the growth of liver metastases in a murine model following the intraportal administration of C3 sarcoma cells. Splenectomy, widely used in murine liver tumor models, has been shown to modify the host’s immune responses (15, 16, 17). We therefore performed direct portal vein injection of tumor cells rather than the conventional splenic injection of tumor cells that requires a secondary splenectomy. We found a significant antitumor effect of FL-based immunotherapy. It is possible that this effect was mediated by several different mechanisms since different populations of the immune effector cells were involved. Our data showed that NK cells played an important role because NK depletion resulted in the inhibition of the antitumor activity of FL. In addition, we have observed an increased infiltration of liver tumors by DC and T lymphocytes, suggesting the possible involvement of DC-mediated stimulation of specific T cell responses. We have also demonstrated an enhanced antitumor effect of the FL/IL-12 combination when compared with the cytokines alone using the described murine tumor model. Thus, these data support the further development of preclinical and clinical studies design to evaluate the efficacy of FL-based immunotherapies, including FL in combination with IL-12 for the treatment of cancer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 (B6, H-2b) mice, 6–8 wk old, were obtained from Taconic (Germantown, NY). The animals were housed in a pathogen-free animal facility, four mice per cage, under controlled temperature and humidity, and a 12-h light/dark cycle with food and water available ad libitum. All animals were acclimatized for at least 2 wk before the initiation of experiments. All experimental procedures were reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
Tumor cell lines
C3 sarcoma is a tumor cell line obtained by tranfecting early mouse embryos with a plasmid containing the entire genome of the human papilloma virus type 16 (18). MC38 is a murine colon adenocarcinoma cell line kindly provided by Dr. S. Rosenberg (Surgery Branch, National Cancer Institute). These tumor cell lines are C57BL/6 syngeneic. They were maintained in RPMI 1640 medium supplemented with 5% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate. Both cell lines were regularly passed through animals and were mycoplasma-free.
Tumor models
Liver metastases were obtained by direct portal injection of MC38 or C3 tumor cells. Mice were anesthetized by methoxyflurane (Mallinckrodt Veterinary, Mundelein, IL) inhalation. The portal vein was exposed through a small midline incision and 300 µl of a suspension of 2 x 105 C3 cells or 1 x 104 MC38 cells in HBSS was injected. Hemostasis was obtained by a gentle compression of the inoculation site with a cotton swab. The animals were sacrificed by cervical dislocation 17 days later. In our preliminary experiments, 17–20 days was found to be the optimal time to count distinct nonconfluent liver metastases in control animals. The spleens and livers were collected and weighed. The liver metastases were counted in a blinded fashion. Blood was either collected by cutting the tail vein for flow cytometry analysis or by direct intracardiac puncture to measure the aminotransferase activity (data presented as international units (IU)) using a Technicon RA-5-00 (Miles, Tarrytown, NY).
Treatment protocols
Mice were randomized and injected with 100 µl i.p. daily for 10 days, starting on day 3 after tumor cell administration of either 0.01% mouse serum albumin (MSA; Sigma, St. Louis, MO) alone (control group) or 10 µg FL in 0.01% MSA. Murine IL-12 (0.2 µg in 0.01% MSA) was administered daily for 5 consecutive days starting on day 10 after tumor cell injection. The animals receiving a combination of FL and IL-12 were injected into both flanks according to the same schedule.
NK depletion
Twenty microliters of anti-Asialo-GM1 antiserum (Wako, Richmond, VA) were injected i.p. every 4 days starting 2 days after the intraportal injection of tumor cells, which is 1 day before FL injections were initiated. Depletion of NK cells was confirmed by FACS analysis of blood and spleen samples.
Morphologic analysis
The entire liver was harvested and examined before and after fixation with neutral buffered formalin (JT Baker, Phillipsburg, NJ) for 4–5 days. From each lobe of the liver, 2–3 slices were routinely processed and embedded in paraffin. Four-micron paraffin sections were stained with hematoxylin/eosin and utilized for pathologic analysis. Morphologic, as well as morphometric analysis of immunohistochemical slides, were performed by an experienced pathologist blinded to sample codes as well as experimental design. All samples were analyzed randomly and at least twice.
Immunohistochemistry
A part of each lobe of the liver was embedded in OCT Compound
(Miles, Elkhart, IN), frozen, and stored at -80°C. Cryostat sections
(6 µm) were used for immunohistochemical evaluation. The following
primary mAbs were used: rat anti-mouse CD8a (Ly-2), rat
anti-mouse CD11b (Mac-1á chain), rat anti-mouse IgG2b
,
rat anti-mouse IgG2a (PharMingen, San Diego, CA), and rat
anti-mouse NLDC-145 (Serotec, Washington DC). Each primary Ab was
applied for 1 h at room temperature. After two washes in PBS, the
sections were incubated with the biotinylated secondary mouse
anti-rat IgG F(ab')2 Ab (Jackson ImmunoResearch,
West Grove, PA) for 45 min followed by an avidin-biotin-peroxidase
complex method using Vectastain ABC kit (Vector Laboratories,
Burlingame, CA). The color reaction was developed for 1–8 min using a
peroxidase chromogen kit (AEC, Biomega, Foster City, CA). Negative
controls included staining with the corresponding isotype for each Ab
and staining with secondary Ab alone. Positive controls included
immunostaining of known positive tissues.
Flow cytometry
The following mAbs were used: phycoerythrin-conjugated rat anti-mouse NK1.1 (clone PK136) and FITC-conjugated rat anti-mouse B220/CD45R Ab (clone RA3-6B2) (PharMingen). The splenic cells were dissociated and filtered through a 70-µm pore size nylon cell strainer; RBCs were lysed, and the remaining cells were stained as previously described (5). After tail vein blood collection in vacutainer tubes containing heparin, the blood samples were diluted with medium, layered over Lympholyte-M (Cedarlane, Hornby, Ontario, Canada), and centrifuged at 200 x g for 30 min. The mononuclear cells (MNC; buffy coat interface) were then harvested and stained as previously described (5).
Reagents
PBS, HBSS, L-glutamine, gentamicin, penicillin, streptomycin, nonessential amino acids, and sodium pyruvate were purchased from Life Technologies (Grand Island, NY). Paraformaldehyde was obtained from Sigma. Human Chinese hamster ovary cell-derived FL was supplied by Immunex R&D Corp. (Seattle, WA). Recombinant murine IL-12 was a gift from Hoffman La-Roche (Nutley, NJ).
Statistical analysis
SigmaStat statistical software package (Jandel Scientific, San
Rafael, CA) was used for data analysis. One-way analysis of variance
(ANOVA) was performed to evaluate the significance of differences
between the experimental groups. All pairwise comparisons of every
combination of group pairs utilized the Student-Newman-Keuls method.
For a single comparison of two groups, the Student’s t test
was applied. To compare the effect of different treatments on liver
metastases number divided into three different categories, the
2 analysis of contingency tables was used. For all
analysis, the level of significance was set at a probability of 0.05 to
be considered significant. Data are presented as means ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the efficacy of FL in the treatment of C3 sarcoma liver
metastases, we treated a total of 19 mice in four independent
experiments (Table I
). The number of
liver metastases was reduced 3-fold in the FL-treated group relative to
the control group. There was no difference in the size of the
metastases between the FL and control groups at gross examination. The
average spleen weight was increased up to 3-fold in the FL-treated
group, although the average liver weight was not significantly changed.
No overt toxicity or weight loss was noted in any of the FL-treated
mice. Liver enzymes (serum glutamic pyruvic transaminases) were lower
in the treated group without reaching statistical significance. These
data suggest that FL therapy has a strong inhibitory effect on the
growth of liver metastases without significant effect on liver
function.
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Livers from both control and FL-treated animals were
analyzed 10 days after the initiation of treatment (Fig. 1). Examination of tissue samples
obtained from the nontreated mice and stained with hematoxylin/eosin
revealed that the tumor-free liver parenchyma had an unmodified
architecture and normal morphology as expected. The border between
metastases and liver parenchyma in the control samples was clearly
indicated, sharp, well demarcated, and characterized by insignificant
MNC infiltration within both tissues. Apoptotic figures were a rare
event within the tumor or the liver parenchyma. In contrast, liver
samples from FL-treated animals showed a significant infiltration of
the border between the metastases and liver parenchyma by two types of
cells. A significant MNC infiltration in association with small
parenchymal necrosis was noted in tumor-free parenchyma. Furthermore, a
marked accumulation of nonparenchymal cells with irregular shaped
nuclei was pronounced at the border of the metastases. MNC infiltration
was also present within the metastases. Importantly, high numbers of
apoptotic figures were found within the liver metastasis. These
observations were also confirmed using FL immunotherapy and another
tumor cell line, MC38, a murine colon adenocarcinoma (data not shown).
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FL enhances the number of DC, macrophages, CD4+, and CD8+ lymphocytes in metastases and liver parenchyma
Livers from both control and treated mice were analyzed 10 days
after initiation of treatment using immunohistochemical techniques.
Examination of livers metastasized by C3 tumors revealed a moderate
increase of CD11b+ (Mac1) cells, presumably macrophages, in
the liver parenchyma and significant increases at the tumor site. We
have also found a moderate but significant increase in both
CD8+ and CD4+ cells in the liver parenchyma and
tumor mass in the samples obtained from the FL-treated animals compared
with the control samples (Fig. 1
). Importantly, FL markedly increased
the number of NLDC-145+ DC in the surrounding parenchyma
and moderately enhanced the number of NLDC-145+ cells
within the liver metastasis (Fig. 1). Analysis of MC38 liver metastases
revealed similar results (data not shown). Thus, we concluded that FL
therapy in mice significantly enhanced the number of DC and macrophages
and to a lesser extent the number of cytotoxic and helper T
lymphocytes, in the liver parenchyma, and within the liver tumor
itself.
NK cells play an important role in the antitumor effect of FL
The percentage of NK cells in the spleens and blood of FL-treated
and nontreated tumor-bearing animals was determined by flow cytometry 1
day after the last administration of FL (day 13). B220 is known to be
expressed on lytically active subsets of lymphokine-activated killer
cells, such as NK cells and non-MHC-restricted CTL. Using this Ab we
wanted to demonstrate that both populations of NK cells,
B220- and B220+ might be affected by the
treatment of mice with FL in vivo. In addition, we and others (4, 5)
have recently demonstrated that murine B lymphocytes were less affected
by the FL administration than T cells, DC, and Gr-1+ cells
in vivo. Based on this observation, we used B220 staining as an
additional control which served to demonstrate the relative specificity
of the FL effect on the generation of NK cells in treated animals. As
shown in Fig. 2, FL therapy markedly
increased the percentage of NK1.1+ cells by 3-fold (8% vs
25%) in the spleen and 2-fold (5% vs 10%) in the blood.
Interestingly, that both populations of NK cells, B220+ and
B220-, were significantly increased, although the increase
in percentage of NK1.1+B220- cell was
substantially higher when compared with the percentage of
NK1.1+B220+ cells. To further evaluate the role
of NK cells in the antitumor effect of FL, we depleted NK cells in the
treated and control animals. NK cell depletion was initiated 2 days
after the portal vein injection of C3 tumor cells (one day before the
initiation of the FL-based therapy). Twenty-two mice in three
independent experiments were randomized into four groups (Fig. 3). We confirmed that FL-treated mice had
fewer metastases than the control mice (45.33 ± 17.69 vs
166.00 ± 22.47, p < 0.05). The NK cell-depleted
and FL-treated mice had significantly more metastases than the
nondepleted ones (160.80 ± 16.02 vs 45.33 ± 17.69,
p < 0.05). The NK cell-depleted mice in the control
group also had more metastases than the nondepleted ones without
reaching statistical significance (176.20 ± 23.80 vs 166.00
± 22.47). There was no difference in the number of liver metastases
between the NK cell-depleted mice in the FL-treated group and the
control group (160.80 ± 16.02 vs 166.00 ± 22.47). Thus,
these data suggest that NK depletion in vivo abrogated the FL-mediated
antitumor effect in a day 3 murine liver tumor model.
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To evaluate the antitumor efficacy of FL/IL-12 combination
therapy, 23 mice received injection of tumor cells in three independent
experiments. As shown in Fig. 4, the
number of hepatic metastases was significantly reduced in the FL- and
FL/IL-12-treated groups compared with the control nontreated group
(19.00 ± 8.29 and 13.52 ± 11.33, respectively, vs
83.43 ± 11.36, p < 0.05) and to the IL-12
monotherapy group (66.00 ± 23.28, p < 0.05).
There were fewer liver metastases in the FL/IL-12 combination group
than in the FL monotherapy group, but the difference did not reach
statistical significance. The reason of this was a single mouse in the
FL/IL-12 group, which developed a large number of metastasis.
Statistical analysis of combined results did not reveal significant
differences between FL and FL/IL-12 groups, although analysis of
individual experiments has demonstrated a statistically significant
difference in two of three experiments (the last one failed because of
a single unusual mouse). To compare the effect of different treatments
on liver metastases number divided into three different categories (see
Table II), we used a 2
analysis of contingency tables. Statistical analysis of these data
revealed that 2 = 239.292 with 6 degrees of freedom,
suggesting that the distribution of the experimental groups within the
chosen categories is significantly different (p
< 0.001). As can be seen from Table II, the percentage of mice with a
limited number of metastases was highest in the FL/IL-12 group. Indeed,
all animals in the FL/IL-12-treated group, except one, had less than 10
metastases. One mouse had 70 metastases, but the metastases were
markedly smaller by gross examination than the metastases found in
other groups. Thus, these results suggest that the FL/IL-12 combination
immunotherapy of liver metastases is more effective treatment than
either one cytokine alone in the established murine liver tumor model.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used direct intraportal injection of C3 sarcoma cells to induce liver metastases. This method was preferred over injection of tumor cells into the spleen or tail vein, because it provided an animal model which is more clinically relevant to study liver metastases. Tail vein injection of tumor cells induces a diffuse metastatic disease that includes overt lung metastases. Splenic injection of tumor cells requires splenectomy which has been shown to be beneficial in tumor-bearing rodents by removing a source of suppressive factors and by eliminating suppressor cell activity (15, 16, 17). Moreover, the spleen has been shown to enlarge due to the extramedullary hematopoiesis during FL administration and may play an important role in FL-based therapy models (5). In addition, a deleterious role of splenectomy has been demonstrated in some human clinical settings of cancer (25). Thus, intraportal administration of tumor cells should serve as a more appropriate way to induce liver metastases to study the relationship between the tumor and immune cells in the liver. FL induces regression of s.c. tumor when administered to animals bearing an Methylcholanthzene-induced fibrosarcoma of B10 origin (7) or C3, MC38, and TS/A tumors (26, 27). Chen et al. (28) have recently reported a similar effect of FL using murine breast cancer cell line C3L5. We have also demonstrated an antitumor efficacy of FL in the murine B16 and CL8-1 melanoma and EL-4 lymphoma models (29). However, there is no information available about the effect of FL immunotherapy during liver metastasis disease.
We showed here that FL had a significant antitumor effect on liver metastases and increased the number of DC and T cells at the tumor site. FL does not have a direct effect on tumor cell lines in vitro (7), but in vivo greatly enhances the number of DC in the spleen and other organs (4, 5). Taken together with the observation that the number of DC at the tumor site positively correlates with a better prognosis of cancers (30), our data support the hypothesis that DC play an important role in the induction of antitumor responses. However, we have recently demonstrated that tumor infiltration with DC may reflect the immunogenicity of murine tumor (29). Because DC play a pivotal role in the induction of immune responses, it is possible to speculate that an increased number of functionally active DC in tumor-bearing host could stimulate a higher level of specific antitumor immune responses. Others have recently made a similar suggestion (31). It is also possible that tumor infiltrating DC play a role in the regulation of T cell survival, including CTL, in the local tumor microenvironment. DC may protect T lymphocytes from tumor-induced apoptotic death (M. T. Lotze, unpublished observation). In fact, we have observed increased number of both CD4+ and CD8+ T cells within liver tumors in FL-treated mice. IL-12 produced by activated DC might, at least in part, be responsible for the alteration of T cell survival by DC.
Furthermore, FL seems to augment the IL-12 production by DC (M. R. Shurin, unpublished data), which additionally supports this hypothesis. The metastases in the FL-treated mice were also infiltrated by lymphocytes, suggesting a role for CTL in the effector phase of antitumor immunity. Indeed we have previously shown that animals that have rejected the s.c. C3 tumor due to the FL treatment, developed specific long-term immune memory and effectively rejected rechallenge with the same tumor cells (26).
We have also shown here that FL induces a strong augmentation of NK
cells in the spleen and blood. Similar results were recently reported
by Shaw et al. (39) who demonstrated that FL treatment sequentially
increased NK activity in the bone marrow, spleen, and liver in normal
mice. NK cells kill target cells without the need for prior
sensitization and without class I MHC restriction. They are also
involved in surveillance and control of hematogenous spread of tumor
cells, which makes them the earliest cellular effector mechanism
against dissemination of metastases (32). Moreover, patients with
advanced metastatic disease often have abnormalities in NK cell
function and/or number (33). The increased number of NK cells in the
spleen of FL-treated animals was still observed 4 days after completing
FL therapy. The percentage of NK cells in the spleens of the animals
bearing liver metastases was higher than previously reported for
tumor-free animals (6). This difference may reflect the effect of
tumor-derived factors on the generation of NK cells, as discussed
above, or the development of antitumor immunity in tumor-bearing mice
at early stages of tumor growth. NK cell depletion significantly
abrogated the antitumor effect of FL in our murine tumor model (Fig. 3), suggesting that NK cell activity was necessary for the inhibition
of tumor growth induced by FL in early therapy model. A similar
mechanism has been observed for the IL-12-based therapy. Early
antitumor effects of IL-12 has been suggested to be mediated by NK
cells while late antitumor effects seem to be mediated by T cells (11).
In fact, the mouse liver contains a large number of NK cells that have
been shown to be potent effector cells against tumors after the
stimulation with IL-12 (34). FL may act similarly to IL-12 or may
stimulate IL-12 production by immune cells, in particular by DC. We
hypothesized that the stimulation of both DC and NK generation and
activity with FL and IL-12 could be a better activator of antitumor
immune responses than either of cytokine alone. In addition, the
interaction between DC and NK cells within the tumor microenvironment
may also play an important role in the effector phase of antitumor
immune responses. We are currently investigating this possibility.
Even though the number of metastases was reduced in the FL-treated animals, there was no significant liver weight loss in this study. On the contrary, there was a trend toward higher liver weights in the FL-treated animals, although it did not reach a statistically significant level. This is in agreement with previously published data (5) demonstrating that in tumor-free animals the liver weight was higher in the FL-treated animals due to newly arrived nonparenchymal cells, a significant number of which were DC. Here, we have confirmed the presence of DC in the livers of tumor-bearing animals.
IL-12 is a cytokine with a potent antitumor activity. It has been shown
that FL synergizes with IL-12 to stimulate the growth and
differentiation of early hematopoietic progenitors in vitro (14). In
this study, we evaluated the antitumor efficacy of FL/IL-12 combination
therapy in the setting of liver metastases in vivo. Our data suggest
that FL/IL-12 therapy was more effective than IL-12 or FL alone for the
treatment of murine liver metastasis. There were more mice with fewer
metastases in the FL/IL-12 group when compared with the FL monotherapy
group (Table II). In fact, only one mouse in this group had more than
10 metastases, although the sizes of these metastases were
significantly smaller than that of the other groups. The antitumor
effect of IL-12 appears to be mediated through early NK stimulation and
later promotion of both CD4- and CD8-mediated responses to tumor (35, 36). The central role of IFN-
in the antitumor effects of IL-12,
promoting the development of a Th1 response, has been demonstrated by
its neutralization (37). IL-12, in the setting of FL administration,
may also promote T cell survival at the tumor site. In addition, IL-12
plays an important role in the mechanism of DC-mediated antitumor
activity. It has been shown that in vivo neutralization of IL-12 in
mice immunized with tumor peptide-pulsed DC blocks the induction of
specific immune response to tumor peptide (38).
In conclusion, we have shown that FL administration induces a significant antitumor effect in a murine C3 liver metastases model and that it is likely mediated by both DC and NK cells. Enhanced antitumor effects of the FL/IL-12 combination were also observed in this tumor model suggesting its consideration for further preclinical and clinical trials.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael R. Shurin, University of Pittsburgh Cancer Institute, Surgical Oncology, Suite 300, Kaufmann Bldg., 3471 5th Avenue, Pittsburgh, PA 15213. E-mail address:
3 Abbreviations used in this paper: FL, FLT3-ligand; DC, dendritic cell(s); MNC, mononuclear cell(s).
Received for publication February 20, 1998. Accepted for publication July 21, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cells with intermediate TCR induced in the liver of mice by IL-12. J. Immunol. 154:4333.[Abstract]
-mediated tumor growth inhibition induced during tumor immunotherapy with rIL-12. Int. Immunol. 8:855.This article has been cited by other articles:
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P. K. Chakravarty, A. Alfieri, E. K. Thomas, V. Beri, K. E. Tanaka, B. Vikram, and C. Guha Flt3-Ligand Administration after Radiation Therapy Prolongs Survival in a Murine Model of Metastatic Lung Cancer Cancer Res., December 1, 1999; 59(24): 6028 - 6032. [Abstract] [Full Text] [PDF]
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J. E. Ohm, M. R. Shurin, C. Esche, M. T. Lotze, D. P. Carbone, and D. I. Gabrilovich Effect of Vascular Endothelial Growth Factor and FLT3 Ligand on Dendritic Cell Generation In Vivo J. Immunol., September 15, 1999; 163(6): 3260 - 3268. [Abstract] [Full Text] [PDF]
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N. S. Williams, J. Klem, I. J. Puzanov, P. V. Sivakumar, M. Bennett, and V. Kumar Differentiation of NK1.1+, Ly49+ NK Cells from flt3+ Multipotent Marrow Progenitor Cells J. Immunol., September 1, 1999; 163(5): 2648 - 2656. [Abstract] [Full Text] [PDF]
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