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Experimental Therapeutics, Preclinical Pharmacology |
-Galactosylceramide (KRN7000) on Spontaneous Hepatic Metastases Requires Endogenous Interleukin 12 in the Liver
Department of Digestive Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
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ABSTRACT |
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-galactosylceramide (KRN7000) on mice
with spontaneous liver metastases of reticulum cell sarcoma M5076 tumor
cells (spontaneous metastasis model). In this model, all mice that were
s.c. challenged with one million tumor cells developed a solid s.c.
mass by day 7 and died of hepatic metastases. In the current study, we
administered 100 µg/kg of KRN7000 to the model mice on days 7, 11,
and 15. This treatment suppressed the growth of established liver
metastases and resulted in the prolongation of survival time.
Fluorescence-activated cell sorter analysis of phenotypes of spleen
cells, hepatic lymphocytes, and regional lymph node cells around the
s.c. tumor revealed that CD3+NK1.1+ (NKT) cells
increased in hepatic lymphocytes of the KRN7000-treated mice. Cytotoxic
activity and IFN-
production of hepatic lymphocytes were augmented
in comparison with those of spleen cells and regional LN cells. At the
same time, interleukin (IL)-12 production of hepatic lymphocytes was
markedly enhanced. Neutralization of IL-12 using a blocking monoclonal
antibody diminished the prolonged survival time. These results showed
that the in vivo antitumor effects of KRN7000 on
spontaneous liver metastases were dependent on the endogenous IL-12
production, where NKT cells in the liver are suggested to be involved.
Adjuvant immunotherapy using KRN7000 could be a promising modality for
the prevention of postoperative liver metastases. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KRN70002
(3)
is a glycosylceramide-containing anomeric
sugar with a long fatty acyl chain (C26) and
sphingosine base (C18) and reported to present
significant antitumor effects on experimental pulmonary metastases of
B6 melanoma in mice because of its strong augmentation of in
vivo NK activity in spleen cells (4)
. These
immunopotentiation and antitumor effects were shown to be attributable
to the antigen-presenting function of DCs, which was activated by
KRN7000 (5)
. DCs are differentiated from monocytes as well
as bone marrow precursors (CD34+ progenitors;
Ref. 6
). DCs possess features that are quite similar to
macrophages, and DCs could take a crucial role in antitumor activity,
because macrophages are one of the effector cells responsible for IL-12
production (7)
. KRN7000 also showed antitumor effects on
experimental hepatic metastasis of Colon26 adenocarcinoma in mice, and
its major antitumor mechanism was reported to be the high activation of
liver-associated NK cells (8)
. Because liver-associated NK
cells take an important role in the antimetastatic activity of
biological response modifiers (8)
, KRN7000 could be
regarded as a biological response modifier. At present, its antitumor
effects on spontaneous hepatic metastases are remained to be
elucidated. The present study examined: (a) antitumor
effects of KRN7000 on s.c. transplanted M5076 sarcoma and subsequent
liver metastases by using the spontaneous liver metastasis model; and
(b) differences of immune responses to KRN7000 treatment
according to the type of effector cells (sources) in major organs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KRN7000.
KRN7000 was generously provided by Dr. Y. Koezuka [Kirin Brewery Co.,
Ltd., Gunma, Japan (3
, 4)
] for this study. The original
solution of KRN7000 (220 µg/ml) was dissolved with 0.9% NaCl
solution containing 0.5% polysorbate 20 (Nikko Chemical, Tokyo,
Japan), and this was diluted by vehicle (0.5% polysorbate 20 in 0.9%
NaCl solution) before use.
M5076 Spontaneous Hepatic Metastasis Model.
The reticulum cell sarcoma M5076 arose as a spontaneous tumor in the
ovary of a C57BL/6 female mouse (9)
. The tumor cells were
isolated from ascites-passaged tumors and cultured in RPMI 1640
supplemented with 17% heat-inactivated equine serum, 100 IU/ml
penicillin, 100 µg/ml streptomycin, and 50 µM of
2-mercaptoethanol in humidified air containing 5%
CO2 at 37°C. M5076 is nonimmunogenic and
preferentially metastasizes to the liver, regardless of the site and
route of tumor injection (9
, 10)
. s.c. injection of 1 x 106 viable M5076 tumor cells to the flank on
day 0 induced spontaneous hepatic metastases in all mice, and all mice
died of liver metastases by day 30. Therefore, this model was used as a
spontaneous hepatic metastasis model.
Antitumor Effect of KRN7000 in Spontaneous Hepatic Metastasis
Model.
Forty mice were challenged s.c. with 1 x
106 M5076 cells to the right flank on day 0.
Twenty mice were administered i.v. with KRN7000 at a dose of 100
µg/kg (in 0.1 ml of vehicle/mouse) on days 7, 11, and 15. Another 20
mice (control) received injections i.v. with 0.1 ml of vehicle on the
same days. For 10 mice each, tumor incidence and growth were evaluated,
and the remaining mice were euthanized on day 19 to weigh the liver. In
addition, the survival of mice challenged with 1 x
106 M5076 cells to the flank and bearing hepatic
metastases was examined in the control and KRN7000-treated groups
(n = 10/group).
Preparation of Spleen Cells, Hepatic Lymphocytes, Regional LN
Cells, and Nonregional LN Cells.
For in vitro experiments to evaluate the immunotherapeutic
properties, spleen cells, hepatic lymphocytes, regional (right
superficial inguinal) LN cells and opposite side (left) LN cells were
obtained on day 19 from the KRN7000-treated mice and control mice.
Spleen cells and LN cells were prepared by forcing the spleen or the LN
pass through a 150-gauge stainless steel mesh, and for spleen cells, in
addition, by treating with RBC lysis solution (0.15
M NH4Cl, 0.1
mM EDTA, and 10 mM
KHCO3). Hepatic lymphocytes were isolated by
density gradient centrifugation using a modification of procedures
described previously (11
, 12)
. Briefly, the liver was
passed through a mesh and suspended in RPMI 1640. After washing,
the pellet was resuspended in RBC lysis solution and washed again. The
cell suspension was overlaid on Lympholyte-M (1.088; Cedarlane,
Ontario, Canada) and centrifuged at 1450 rpm for 20 min at room
temperature. The interface was aspirated, washed twice, and subjected
to in vitro assays.
Immunophenotypic Analysis.
Surface phenotypes of the cells were identified by using mAbs in
conjugation with two-color immunofluorescence test. The mAbs used
included FITC antimouse CD3, FITC antimouse CD4, PE antimouse CD8, and
PE antimouse NK1.1 (PharMingen, San Diego, CA). The presence of
fluorescence-positive cells was analyzed by FACS Calibur (Becton
Dickinson, Mountain View, CA).
Cytotoxicity Assay.
The target cells (M5076, YAC-1, and MC-38 tumors) were labeled with 100
µCi (per 2 x 106 cells) of
Na251CrO4
for 60 min at 37°C in complete medium and washed twice with the
medium. Labeled targets (5 x 103
cells/well) were incubated in a total volume of 200 µl with effector
cells in 10% FCS-RPMI 1640 in 96-well, roundbottomed microtiter
plates. After 16 h of incubation, the supernatant was harvested
and counted in a gamma counter. Cytotoxicity was calculated as the
percentage of releasable counts after subtraction of the spontaneous
release.
IFN- and IL-12 Productions.
Spleen cells, hepatic lymphocytes, and LN cells were suspended to 10%
FCS-RPMI 1640 medium at a density of 5 x
106 cells/ml and irradiated (25 Gy) M5076, YAC-1,
or MC-38 cells were suspended to the medium at a density of 2 x
105 cells/ml. Then 0.5 ml each of the lymphocytes
and the tumor cells were poured to 48-well plates (total, 1 ml/plate)
for lymphocyte stimulation. Supernatants harvested after 48-h
incubation in a humidified atmosphere of 5% CO2
in air at 37°C were tested for IFN- and IL-12 using ELISA kits
(Genzyme, Cambridge, MA).
M5076 Residual Hepatic Metastasis Model.
In the above-mentioned in vivo experiment system, survival
time prolongation of the tumor-bearing mice was difficult because s.c.
tumor (primary tumor) was remained, and KRN7000 was ineffective to
these tumors. However, KRN7000 was expected to be efficacious to
residual hepatic metastases because it was sufficiently effective on
spontaneous liver metastases. On the other hand, because residual
hepatic metastases are encountered frequently in clinical practices,
its clinical efficacy on residual hepatic metastases was thought to be
worthwhile to examine.
Almost all mice inoculated with 1 x 106 M5076 cells to the right flank on day 0 died of spontaneously metastasized hepatic tumors, although the established s.c. tumor was surgically removed under ether anesthesia on day 7. Therefore, this model was used as the residual hepatic metastasis model. Consequently, after the removal of primary tumor on day 7, mice were administered i.v. with 100 µg/kg of KRN7000 on days 7, 11, and 15, and their survival times were examined.
Neutralization of IL-12 in Vivo.
To clarify the involvement of endogenous IL-12 to antitumor effects on
liver metastases, IL-12 was neutralized in vivo by
administering anti-IL-12 mAb (13)
. In brief, 100
µg/mouse of IL-12 neutralizing mAb (C17.8) or isotype-matched control
mAb (R35–95; PharMingen, San Diego, CA) was administered i.p. at
6 h before the KRN7000 administration on days 7, 11, and 15, and
the survival time was monitored. Neutralization of IL-12 activity was
confirmed by ELISA analysis of serum samples (data not shown).
Statistical Analysis.
Animals’ survival time was compared with the Kaplan-Meier estimates,
and significance was determined by the generalized Wilcoxon test. ANOVA
and Student’s t test were used to analyze the other data.
Data of in vitro assays were cumulated from three
independent experiments, each performed with pools of samples from four
mice, and values were expressed as means ± SE.
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RESULTS |
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3 mm in diameter. Starting from day 7, the
mice received i.v. injection of either 0.1 ml of vehicle or KRN7000
(100 µg/kg in 0.1 ml vehicle) on days 7, 11, and 15. Growth of the
established s.c. tumor was not suppressed by the treatment (Fig. 1
A), whereas the growth of liver metastases measured in terms
of liver weight on day 19 was significantly suppressed in the
KRN7000-treated mice, i.e., 1.19 ± 0.03 g in the
treated mice, 1.99 ± 0.21 g in the nontreated mice
(P = 0.004), and 1.14 ± 0.05 g in
age-matched normal mice (Fig. 1
C). In addition, there were
no macroscopically detectable liver metastases in the liver of
KRN7000-treated mice. The treatment also prolonged survival time
significantly (P = 0.014; Fig. 1
B).
Antitumor effect of KRN7000 was remarkable in spontaneous hepatic
metastases, although it was not apparent in the established s.c.
tumors.
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. In hepatic lymphocytes, phenotypic changes between CD4 and CD8 was
not observed, but the ratio of the cells that were positive to both CD3
and NK1.1 increased in the KRN7000-treated mice, i.e.,
22.3 ± 0.9% in the treated mice and 14.6 ± 2.1% in the
nontreated mice (P = 0.028; Fig. 2
).
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,A and B). The lytic activity to MC-38 was low in
the all lymphocytes examined (Fig. 3
, E and F).
However, the lytic activity of hepatic lymphocytes to YAC-1 increased
significantly in the KRN7000-treated mice, i.e., 45.1 ± 4.9% in the treated mice and 9.8 ± 1.5% in the nontreated
mice (E:T, 80; P < 0.001; Fig. 3
, C and
D). These findings indicate that the efficacious antitumor
effect of KRN7000 against liver metastases may be attributable mainly
to the enhanced NK activity of hepatic lymphocytes.
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and IL-12 Productions by KRN7000. production in cocultures
of spleen cells or LN cells with M5076 was low and at a similar level,
whereas IFN- production in the coculture of hepatic lymphocytes
increased significantly in the treated mice (111.9 ± 47.4 pg/ml)
from the level of the nontreated mice (6.3 ± 2.1 pg/ml;
P = 0.020; Fig. 4
A). In the cocultures with YAC-1, IFN- productions in the
three types of lymphocytes from nontreated mice were relatively higher
than those in the cocultures with the other tumor cells, but the
increase of IFN- in the KRN7000-treated mice was significant only in
hepatic lymphocytes, i.e., 176.7 ± 77.9 pg/ml in the treated mice and 41.0 ± 13.0 pg/ml in the nontreated
mice (P = 0.022; Fig. 4
B), and this increase
was larger than that in the coculture with M5076 (Fig. 4
A).
In the coculture with MC-38, IFN- productions of the three
lymphocytes were low and at a similar level to those in cultures
without any tumor cells, regardless of KRN7000 treatment (Fig. 4
,
C and D).
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shows that IL-12 production in KRN7000-treated hepatic lymphocytes was
quite high, not only in the cocultures with M5076, YAC-1, or MC-38, but
also in the cultures without tumor cells, and these production levels
did not differ significantly. On the other hand, IL-12 productions in
the spleen cells, regional LN cells, and hepatic lymphocytes without
KRN7000 treatment, as well as the productions in KRN7000-treated spleen
cells and regional LN cells, were low and at a similar level.
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A). In addition, 11.1% of the treated mice had survived
until day 100, whereas there were no surviving mice in the nontreated
group on that day.
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level also decreased significantly from 49.1 ± 4.4 pg/ml
(control mAb) to 10.0 ± 6.9 pg/ml (anti-IL-12 antibody;
P = 0.041). On day 19 of the mice that received
anti-IL-12 antibody, cytotoxicity of hepatic lymphocytes against
NK-sensitive YAC-1 was 12.8 ± 2.6%, the ratio of NKT cells was
15.2 ± 3.4%, and liver weight was 2.13 ± 0.27 g,
whereas these data in the mice with control mAb on day 19 were
43.6 ± 5.2%, 21.7 ± 1.1%, and 1.22 ± 0.15 g,
respectively. When anti-IL-12 mAb was administered, survival times of
mice that received KRN7000 treatment was not prolonged
(P = 0.033; Fig. 6
B). |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
To clarify the mechanism of effectiveness on liver metastases,
differences in phenotypes and immune responses were examined in:
(a) spleen cells as an indicator of systemic immunity;
(b) hepatic lymphocytes as an effector in the liver;
(c) regional LN cells as an effector in the area proximal to
the tumor; and (d) nonregional LN cells as a control
effector to the regional LN cells. FACS analysis showed that:
(a) NKT cells were more abundantly present in hepatic
lymphocytes than in spleen cells (17)
; and (b)
this larger number of NKT cells in hepatic lymphocytes increased
further with KRN7000 treatment (Fig. 2)
. In addition, enhanced
cytotoxic activity of hepatic lymphocytes, which was suggested to be
mainly attributable to the increased in vivo NK activity
(8)
, was shown in comparison to the other effector cells
(Fig. 3)
. IFN- production in cocultures of hepatic lymphocytes
with tumor cells increased in KRN7000-treated mice (Fig. 4)
, and the
increase was the highest with YAC-1 and then M5076, followed by MC-38.
However, IL-12 levels were high, regardless of the cultures with or
without tumor cells, and the production was specific to the derived
organ (liver) of the effector (hepatic lymphocytes; Fig. 5
). On the
other hand, KRN7000 was ineffective to s.c. transplanted tumor,
although it was effective on spontaneous liver metastases. The possible
explanations for this are: (a) the regional LN cells and
spleen cells consist of almost no NKT cells (Fig. 2)
; and
(b) their IFN- and IL-12 productions were low (Figs. 4
and 5)
.
DCs are differentiated from monocytes as well as
CD34+ progenitors (6)
. DCs possess
features similar to macrophages, which are one type of the effector
cells responsible for producing IL-12 (7)
. In addition,
DCs or their progenitors can be propagated from parenchymal organs such
as the liver (18
, 19)
. As a process of antitumor effect of
KRN7000, Kawano et al. (14)
reported that
KRN7000 was presented by CD1d molecules expressed on DCs, and the
KRN7000/CD1 complex together with CD40/CD40 ligand (CD40L) and B7/CD28
interaction selectively stimulated the proliferation of NKT cells.
Kitamura et al. (16)
demonstrated that NKT
cells recognized KRN7000-bound DCs via their T cell receptors,
interacted with DCs via CD40/CD40L, and then the DCs produced IL-12.
This endogenously produced IL-12 stimulated NKT cells to produce
IFN-, and this IFN- up-regulated IL-12 receptors on NKT cells in
an autocrine manner. In addition, Kawano et al.
(15)
reported that the effector function of
KRN7000-activated NKT cells is the lysis of target tumor via a T-cell
receptor-independent, NK-like nonspecific mechanism with perforin.
Therefore, in antitumor effects of KRN7000 in the liver, not only NKT
cells but also DCs are thought to take an important role. In the
present study, IL-12 production of the hepatic lymphocytes in
KRN7000-treated mice increased without coculturing with tumor cells.
This increase might be mediated by hepatic DCs or their progenitors
that are already present in the liver.
NKT cells were also reported as a primary target of IL-12, and
the cells exert a major effector function in IL-12-mediated tumor
rejection (20)
. We blocked in vivo IL-12
activity with neutralizing mAb administration, and the findings
suggested that the increase of IL-12 production in hepatic lymphocytes
of KRN7000-treated mice affects: (a) the increase of IFN-
production attributable to M5076 or YAC-1 stimulation (Fig. 4)
; and
(b) the enhancement of cytotoxic activity against these
target tumor cells (Fig. 3)
. However, in the coculture with another
syngeneic tumor MC-38, or in the cultures without tumor cell
stimulation, IFN- production did not increase nor was the cytotoxic
activity enhanced, although IL-12 production of hepatic lymphocytes
increased because of KRN7000 treatment (Fig. 5)
. In this way, the
present study was unable to elucidate the detailed mechanism of the
increase of IFN- production and the enhancement of cytotoxic
activity attributable to the increase of IL-12 production. Endogenously
produced IL-12 was not a sufficient condition, but this could be a
necessary condition to the antitumor effects of KRN7000 treatment.
Kitamura et al. (16)
reported that endogenous
IL-12 production by DCs was necessary in triggering NKT cells in their
in vitro blocking study to IL-12 using anti-IL-12 mAb, and
they also showed that IL-12 enhanced expression of IL-12R mRNA in
spleen cells in another in vivo IL-12 blocking experiment.
However, the mechanism of in vivo immunological activation
of KRN7000 has remained unknown, and the necessity of endogenously
produced IL-12 in antitumor effects of KRN7000 on spontaneous
metastases, which are frequently encountered in clinical practices, has
also been remained unclear. The present study, therefore, neutralized
IL-12 in vivo using anti-IL-12 mAb and examined the decrease
of antitumor effect of KRN7000. As a result, when anti-IL-12
neutralizing mAb was administered at the KRN7000 treatment to the
residual hepatic metastasis model, survival times of the mice were not
prolonged (Fig. 6
B). This showed that in vivo
neutralization of IL-12 activity diminished antitumor effect of
KRN7000, and this then showed the presence of endogenously produced
IL-12 as a necessary condition for the antitumor effect of KRN7000.
KRN7000 showed antitumor effect to mice with liver metastases, but the
effect was insufficient from the viewpoint of cure (Fig. 6
A). Kawano et al. (14)
proposed
that the primary mechanism of antitumor effect of KRN7000 is its
binding to DCs, which selectively stimulates NKT cells. Another
study reported that FLT3-ligand, a cytokine that stimulates
proliferation and differentiation of hematopoietic progenitors,
enhanced the number of DCs in the liver parenchyma and within the liver
metastases (21)
. These findings propose that
coadministration of KRN7000 with DCs or a means that differentiate or
proliferate DCs would magnify clinical efficacy of KRN7000. In
addition, KRN7000 would be one of the most promising adjuvants in
cancer vaccine therapy using DCs and tumor antigens, because KRN7000 is
known to have enhancing effects to the antigen-presenting function of
DCs (5)
. On the other hand, it is unknown whether KRN7000
is effective against metastasis in the organs other than the liver. One
possible effective organ with metastases is the lung, where NKT cells
are reported to be present (22)
. It would also be
worthwhile to examine the other administration methods, such as
coadministration of KRN7000 with DCs and/or NKT cells, in the treatment
of metastases in organs other than the liver and lung. In addition,
IL-12 has antiangiogenic activity as well as being an activating
cytokine for NKT cells (23)
. From the viewpoint of the
activation of tumor immunity, further studies are necessary to examine
the antitumor activity of KRN7000.
KRN7000 was also reported to stimulate human NKT cells in a CD1d-dependent manner (24 , 25) . Immunotherapy using KRN7000 is expected to be a useful adjuvant modality in treatment for postoperative residual cancer in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Digestive Surgery, Kyoto Prefectural
University of Medicine, 465 Kawaramachi Hirokoji, Kamigyo-ku, Kyoto
602-8566, Japan. Phone: 81-75-251-5527; Fax: 81-75-251-5522. 
2 The abbreviations used are: KRN7000,
(2S,3S,4R)-1-O-(-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol;
DC, dendritic cell; IL, interleukin; NK, natural killer; NKT, natural
killer T; LN, lymph node; mAb, monoclonal antibody.
Received 11/15/99; revised 4/24/00; accepted 4/24/00.
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  Y.-J. Chang, J.-R. Huang, Y.-C. Tsai, J.-T. Hung, D. Wu, M. Fujio, C.-H. Wong, and A. L. Yu Potent immune-modulating and anticancer effects of NKT cell stimulatory glycolipids PNAS, June 19, 2007; 104(25): 10299 - 10304. [Abstract] [Full Text] [PDF]
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 J. W. Molling, J. A.E. Langius, J. A. Langendijk, C. R. Leemans, H. J. Bontkes, H. J.J. van der Vliet, B. M. E. von Blomberg, R. J. Scheper, and A. J.M. van den Eertwegh Low Levels of Circulating Invariant Natural Killer T Cells Predict Poor Clinical Outcome in Patients With Head and Neck Squamous Cell Carcinoma J. Clin. Oncol., March 1, 2007; 25(7): 862 - 868. [Abstract] [Full Text] [PDF]
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 M. J. Smyth, M. E. Wallace, S. L. Nutt, H. Yagita, D. I. Godfrey, and Y. Hayakawa Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer J. Exp. Med., June 20, 2005; 201(12): 1973 - 1985. [Abstract] [Full Text] [PDF]
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Y. Hayakawa, S. Rovero, G. Forni, and M. J. Smyth {alpha}-Galactosylceramide (KRN7000) suppression of chemical- and oncogene-dependent carcinogenesis PNAS, August 5, 2003; 100(16): 9464 - 9469. [Abstract] [Full Text] [PDF]
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) or vehicle only (
or 
) on days 7, 11, and 15.
A, antitumor effect was not observed on the s.c.
established tumor. B, survival time of KRN7000-treated
mice was prolonged and became significantly longer than that of
nontreated mice (P = 0.014). C,
liver metastasis was evaluated in terms of liver weight. On day 19,
liver weights of KRN7000-treated mice were significantly lower than the
control mice (P = 0.004). Each experiment was
performed at least three times, and the results of a representative
experiment were presented; bars, SE. 
