Download Maltose Tetrapalmitate, a Nontoxic

[CANCER RESEARCH 38, 3315-3321, October 1978]
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Maltose Tetrapalmitate, a Nontoxic immunopotentiator with Antitumor
Activity1
Vijai N. N ¡gam, Carlos A. Brailovsky, ' and C. Chopra
Départementd'Anatomie et de Biologie Cellulaire, Facultéde Médecine,Universitéde Sherbrooke, Sherbrooke, Québec,Canada, J1H 5N4
ABSTRACT
The attempt to synthesize a lipid A-like component (the
active portion of lipopolysaccharides, but lacking its endotoxic activity) resulted in the production of fatty acyl
sugars of which maltose tetrapalmitate was seen to yield
the most promising results. It shows no endotoxic activity
and elicits an antitumor response in tumor-transplanted
animals as shown by (a) an enhancement of the host's
capacity to reject a large number of tumor cells, (b)
retardation of growth in tumor size, and (c) induction of
hemorrhagic necrosis in certain tumors. Experiments with
mammary ascites carcinoma show maltose tetrapalmitate
to be as effective as is bacterial glycolipid mR595 in its
antitumor activity. The degree of sensitivity to maltose
tetrapalmitate varies with the tumor-host system: mam
mary ascites carcinoma < NH = CI2TSV,S = B16 < L26.
The mode of action of maltose tetrapalmitate appears to
be via its modulation of the immune system. It is itself
noncytotoxic to tumor cells in vitro. It is seen to stimulate
the spleen cells of certain animals mitogenically, although
it causes tumor rejection in all the types of animals tested.
Also, it activates peritoneal exúdate macrophages in
tumor-bearing animals; whether specifically or nonspecifically has not yet been established.
INTRODUCTION
It is well known that endotoxic bacterial LPS" possess
immunological reactivities such as mitogenic stimulation of
B-lymphocytes (2), macrophage activation (1), replacement
of helper T-cell function during antibody production against
antigens in vivo (15), and conversion of a tolerogenic dose
of an antigen to an immunogenic dose (8). In high doses
LPS are also known to be immunosuppressive (9). The
former features (1, 2, 8, 15) of the LPS molecule make them
useful substances in the immune rejection of tumors. How
ever, their endotoxic activity prevents their application in
human immunotherapy despite their known antitumor po
tential against certain transplanted tumors «10,11). Lipid A
is generally considered to be the active part of the LPS
molecule that elicits immunological reactivity and pos
sesses endotoxic activity.
Our purpose in this study was to synthesize a lipid A-like
molecule that would be immunologically reactive and de1 Supported by National Cancer Institute of Canada and Ministère de
l'Education du Québec.
2 Work done during the tenure of a Research Associateship of the National
Cancer Institute of Canada.
3 Chercheur Boursier of the Quebec Medical Research Council.
4 The abbreviations used are: LPS. lipopolysaccharides, SRBC, sheep red
blood cells; MTP, maltose tetrapalmitate; MAC, mammary ascites carcinoma;
RPMI, Roswell Park Memorial Institute; PFC, plaque-forming cells; PEC,
peritoneal exúdatecells; ¡.t.,¡ntratumorally;BCG, Bacillus Calmette-Guérin.
Received December 15, 1977; accepted June 26, 1978.
OCTOBER
void of endotoxic activity so that it could be tested for its
antitumor activity. Antitumor activity in such a molecule
would suggest that (a) enhancement of immune response
can cause tumor rejection and (b) endotoxic activity need
not accompany immune reactivity or tumor rejection ability.
During the past 3 years we have synthesized several
glycolipid esters by fatty acylation of simple carbohydrates
and nucleosides. These are termed "synthetic glycolipids."
Those derived by O-esterification of maltose, cellobiose,
and D-galactose with palmitoyl chloride were found to
enhance serum antibody titers eight-fold in rabbits against
SRBC used as an antigen (unpublished data). They were
also mitogenic for spleen lymphocytes of Swiss mice in
vitro. One of the glycolipids derived from maltose has been
tentatively characterized as MTP and has been subjected to
investigation for its antitumor activity, for its ability to act as
an immunopotentiator, and for its toxicity. This paper will
describe the results of this investigation and, briefly, the
synthesis of this compound.
MATERIALS
AND METHODS
Synthesis of MTP. Dry maltose (10 mmol) was dissolved
in 40 ml of dry dimethylformamide at 60°,and 5 ml of dry
pyridine were then added. To the mixture a solution of
palmitoyl chloride (40 mmol) in 5 ml dry dimethylformamide
was added dropwise with stirring. The mixture was left at
60°for 4 to 5 hr and then left at room temperature. It was
subjected to thin-layer chromatography on Silica Gel G
plates with CHCI:,:CH,OH:HoO (60:25:4) as the developing
solvent. The plates were stained with resorcinol reagent
(20). Six bands with R,,'s of 0.95, 0.80, 0.68, 0.54, 0.39, and
0.23 were identified. The RK 0.68 band occurred in the
largest amount. This glycolipid was isolated by chromatog
raphy on a Silica Gel G column (3.8 x 40 cm) with the
solvent mixture described above. A yield of 200 to 300 mg
was obtained. On hydrolysis (4 N MCI for 4 hr at 100°),it
gave a mixture of glucose and palmitic acid in the approxi
mate ratio of 1:2, both determined colorimetrically (5, 18).
Carbon and hydrogen analyses gave the following result for
MTP.
Calculated:
Found:
C 70.5, H 11. 09
C 69.92, H 11 .15
The compound was nonreducing, indicating that C-1 was
esterified. Periodate oxidation of the compound followed
by sodium borohydride reduction and mild acid hydrolysis
(18) failed to reveal the presence of glucose upon paper
Chromatographie analysis of the reaction mixture. This
indicates that both glucose residues of maltose contain a
pair of adjacent hydroxyl groups in the MTP molecule. The
1978
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3315
V. N. Nigam et al.
compound is suggested to be either 1,6,2',6'- or 1,6,4',6'tetra-O-palmitoyl
maltose.
Tumors and Antitumor Activity Determination. The transpiantabile tumors used for the determination
of antitumor
activity of MTP, with their source given in parentheses,
were as follows: MAC 13762 in Fischer 344/CRBL rats (Dr.
A. E. Bogden, Mason Research Institute, Worcester, Mass.);
ascites form of Novikoff hepatoma in Sprague-Dawley
rats
(Dr. R. Moráis, Institut du Cancer de Montréal, Montreal,
Canada); cultures of a SV40-induced sarcoma (CI2TSV.-,S) in
Syrian hamsters (Dr. P. Tournier, Centre National de Re
cherche Scientifique,
Villejuif, France); line 26 colon tumor
(L26) in BALB/c mice (Dr. T. H. Corbett, Southern Research
Institute, Birmingham,
Ala.,); and B16 melanoma (B16) in
C57BL/J
mice (The Jackson
Laboratory,
Bar Harbor,
Maine). The required tumor recipients were purchased from
Charles River Breeding Co., St. Constant, Quebec, Canada,
and from The Jackson Laboratory.
A known number of tumor cells or a piece of tumor of
known size was transplanted
s.c. above the right thigh of
the animals on Day 0, and animals were treated with 10 /ug
MTP suspension in 0.3 ml 0.9% NaCI solution per animal
s.c. on the left thigh on Day 1 and then every third day until
the termination of the experiment. Controls received 0.9%
NaCI solution alone. The number of animals with and
without tumor and the tumor size were recorded. In other
experiments tumors were allowed to grow to approximately
1 cu cm, and MTP treatment was begun and continued as
described previously.
Toxicity Determination. The toxicity of MTP was deter
mined in the chick embryo lethality test (16) and the endotoxic activity by the Limulus lysate assay (7). Groups of
Swiss mice were also treated i.p. with an increasing dose of
MTP up to 10 mg/mouse, and the number of animals that
died within 48 hr were recorded.
Immunological Techniques. Isolation of spleen lympho
cytes and their stimulation
by MTP were carried out by
techniques similar to those described by Weinstein ef al.
(21). RPMI Medium 1640 containing
10% heat-inactivated
fetal bovine serum was used throughout the investigation.
PFC were determined according to the method of Jerne ef
al. (6), 5 days after an i.p. injection of sheep RBC was given
to rats. Serum anti-sheep RBC antibody titers were deter
mined according to Beckmann ef al. (3), 10 days after 10"
sheep RBC injection i.p. was given to rats. Antibody titers
against tumor cells were determined by the complementfixation technique (12).
PEC were isolated by lavage of the peritoneal cavity with
phosphate-buffered
saline, 2 days after a 10-ml injection of
mineral oil to rats had been given. Washed oil-free PEC
were suspended in RPMI Medium 1640 containing 10% fetal
bovine serum. For in vitro activation of PEC by MTP, 106
PEC in 0.2 ml medium were added to a number of microtiter
plates, one-half of which contained 5 /¿9MTP in 10 M10-9%
NaCI solution. They were incubated for 48 hr in a CO2
incubator
(95% air-5% CO2). The medium was then re
moved, and to the attached cells 10" MAC cells in 0.2 ml
medium
were
added.
After
3 days of incubation
[3H]thymidine (1 mCi in 10 /¿I)was added to all the wells,
and 18 hr later cellular DMA was precipitated by acid and
counted for its 3H. Controls consisted of untreated and
3316
MTP-treated PEC and MAC alone. Percentage of cytotoxicity was used as a measure of in vitro stimulation of PEC by
MTP and was given by
Fcpm in MAC-PEC
100
|
- cpm in PEC
x 100~j
cpm in MAC
For determination
of MTP-mediated in vivo activation of
PEC and spleen lymphocytes that could render them cytotoxic to injected tumor (MAC or CLTSV.-.S) cells, 4 groups of
animals (rats or hamsters) each were treated with 0.5 ml
0.9% NaCI solution i.p., 10 /ng MTP i.p., and 106 tumor cells
s.c. The animals received injections i.p. of 10 ml mineral oil
4 days later. After 3 days animals were sacrificed and their
PEC and spleen cells were isolated. Cytotoxicities
of rat
and hamster spleen lymphocytes and PEC against target
MAC and CUTSV.-.S, respectively, were determined. An un
related tumor cell line (spontaneously
transformed Wistar
rat fibroblasts)
was used as a nonspecific
tumor target
described against rat PEC.
Materials. Maltose was purchased from Sigma Chemical
Co., St. Louis, Mo., and palmitoyl chloride was from East
man Kodak, Rochester, N. Y. All other chemicals were
obtained from Fisher Scientific
Co., Montreal, Canada.
RPMI Medium 1640 and fetal bovine serum were purchased
from Flow Laboratories, Rockville, Md. Bacterial glycolipid
mR595 was a gift from Dr. Otto Luderitz, Max-Planck
Institut fürImmunobiologie,
mice of BALB/c background
tal animal house facilities.
Freiburg, West Germany. Nude
were raised in our departmen
RESULTS
Toxicity of MTP. LD.->(,
of MTP in Swiss mice could not be
determined because quantities were limited and amounts
of MTP as large as 10 mg/mouse
injected i.p. failed to
cause mortality of even 1 mouse in a group of 10. Bacterial
endotoxin (mR595) gave a dose lethal to 50% of mice of
400 fj.g in the same test. Similarly, in the chick embryo
lethality test, 100 ¿¿g
of MTP were nontoxic. The compara
tive figure for the dose lethal to 50% mice of mR595 was
0.007 /¿g.In the Limulus lysate assay, 100 ^g MTP per ml
failed to induce gel formation,
whereas 1 mg MTP/ml
caused a thickening
of the lysate. In this assay mR595
caused gel formation at a concentration
of 0.001 /¿g/ml.
Antitumor Activity of MTP against Transplanted Tumors.
MTP was tested for its antitumor activity in 5 transplanted
animal tumor systems in 3 species of animals, as described
in "Materials and Methods." In addition the effect on the
growth of CI,TSV5S in nude mice by MTP was also tried. An
optimum MTP dose of 10 ¿ig/amma! was selected for all
experiments after a preliminary screening of the dose-re
sponse relationship with 2 tumor-host systems (MAC and
CI2TSV.-,S) indicated that, although a dose as low as 0.1 ¿¿g
MTP per animal sometimes retarded increase in tumor size,
consistent results were obtained with 10 /ng per animal.
Subsequent work confirmed this finding (see later). The
results of the experiment
are shown in Table 1. It was
observed that insofar as tumor development
by a known
and limited number of inoculated tumor cells was con
cerned, MTP was effective in reducing the number of tumor
takes in all of the 3 tumors used (MAC, NH, and CI2TSV,S).
CANCER
RESEARCH
VOL. 38
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Antitumor Activity of MTP
Table 1
Antitumor activity of MTP against transplanted tumors
For details, see "Materials and Methods."
of cells
transplanted
or tumor
size5x
1035
1035x
x
1035
1031
x
10«1
x
10"1
x
Tumor
typeMACNHCI2TSV5SEx-perimentFischer
Recipient
123Sprague-Dawley
344 rats
1rats2Syrian
hamsters
1061
x
10«1
x
10«1
x
10"1
x
solutionMTP0.9%
NaCI
0.2)"'r2/5
(2.3 ±
0.1)6'"0/1
(1.9 ±
solutionMTP0.9%
NaCI
±0.2)2/10(1
0(3.2
0.2)0/5 .9 ±
0.3)0/5(3.2 ±
±0.3)"1/9
(2.9
solutionMTP0.9%
NaCI
solutionMTP0.9%
NaCI
solutionMTP0.9%
NaCI
x
123No. 10"1
solutionMTP0.9%
NaCI
1061
x
10s1
x
solutionMTP0.9%
NaCI
10"1
x
1071
x
solutionMTPAnimals
NaCI
x 107Treatment0.9%
±0.5)"8/24(2.1
(4.1
0.3)61/9 ±
0.4)68/9
(4.0 ±
(2.6±0.3)62/10(2.0
NaCI solution
MTP0.9%
(2.0 ±0.1)
11/12(1
.S/0/4
2
10"1
8
8
1
1
NaCI solution
MTP
0.9% NaCI solution
MTP0.9%
1/4 (2.0 ±0.2)
0/10(2.9 ±0.4)
±0.3)0/6
2/10 (1.8
NaCI solution
MTP1/4
(6.0 ±0.4)
1/10(3.5 ±0.3)NDPNDND
cu mm
cu mm
x 10s
x
1 x 106
1 x 10"0.9%
5.931.6
±
4.7d28.5
±
5.645.1
±
6.2"62.2
±
6.584.0
±
±7.2"
0.2)7/10(1.1±
0.2)0/105/10Survival
±
8 cu mm
8 cu
miceNude
times of
animals with es
tablished tumor
(days)23.9
0.5)10/24
(3.9 ±
±0.2)4/9
(1.8
mm1
L26B16CI2TSV5SBALB/c
miceC57BL/J
mice1
without tumor/
total no. of animals at
days0/5
21 -24
(2.5 ±0.3)'IJ
" Numbers in parentheses, tumor size (sq cm).
6 Antibody titers in tumor-bearing animals were as follows: Anti-MAC, 0.9% NaCI solution-treated 1 to 1/2, MTP-treated 1/2 to 1/4
without tumor 1/4 to 1/16; Anti-NH, 0.9% NaCI solution-treated 1/2 to 1/16, MTP-treated 1/16 to 1/32, without tumor 1/64 to 1/1024; antiCI2TSV5S,0.9% NaCI solution-treated 1/2 to 1/4, MTP-treated 1/4 to 1/8.
c Mean ±S.D.
d The differences between untreated and MTP-treated animals are statistically nonsignificant.
e ND, not done.
^Tumors were hemorrhagically necrotized.
Moreover, the size of tumors that developed in MTP-treated
animals was smaller than that of 0.9% NaCI solution-treated
controls. Among these 3 tumors MAC was the least sensitive
to MTP treatment, a 2-fold increase in the number of tumor
cells injected (from 5 x 103 to 1 x 10* cells) abolished the
ability of MTP to induce rejection of tumor cells, and the
tumors that developed were of the same size as those
observed in the control animals. In contrast to MAC MTP
was much more effective against CI2TSVr>Stumor in ham
sters. The viable cell inoculum for 50% tumor takes given 4
and 5 weeks after tumor inoculation, was found to be 2 x
103 cells in the case of 0.9% NaCI solution-treated control
animals and 1 x 107cells in the case of animals treated with
MTP (Chart 1).Thus, MTP treatment enhanced the ability of
the animals to reject a 5000-fold higher tumor cell dose.
The solid tumors (L26 and B16) transplanted s.c. via
trocar were also sensitive to MTP-induced tumor rejection.
Thus, whereas 1 of 4 animals transplanted with L26 rejected
the tumor transplant on 0.9% NaCI solution treatment, 11 of
12 rejected tumor on MTP treatment. In the case of B16,
MTP treatment resulted in the rejection of the tumor in
about 20% of the mice in 2 experiments compared to no
OCTOBER
rejection in animals kept on 0.9% NaCI solution treatment.
In both of these cases, the animals that failed to reject the
tumor, those treated with MTP bore smaller tumors than did
0.9% NaCI solution-treated controls.
The effect of MTP treatment on the survival times of
animals that bear growing MAC, NH, and CI2TSV.-,Stumors
is shown in Table 1. Treatment began when the tumor had
reached a size of approximately 1 cu cm. It was observed
that MTP did not cause rejection of these large tumors and
that the survival times of animals bearing MAC, NH, and
CI2TSV.-,Stumors were insignificantly increased.
When nude mice were given transplants of a xenograft of
CI2TSV5Scells and MTP treatment was instituted, the tumor
cells were not rejected but the size of tumore that developed
was smaller in the MTP-treated group than in the 0.9% NaCI
solution-treated controls (Table 1).
Since MAC was the least sensitive to s.c. MTP injection
away from the tumor site, we tried i.t. injection of MTP in
MAC-bearing animals. Neither a rejection of the tumor nor
a decrease in tumor size was observed. Further, when MTP
was injected on Day -7, -3, and 0 and the animals were
inoculated with 10s MAC, the tumor cells were not rejected,
1978
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3317
V. N. Nigam et al.
and neither was the size of tumors that developed in MTPpretreated animals smaller than in the controls.
MTP Dose and Antitumor Response Relationship. Chart
2 shows the number of tumor takes and the size of tumors
that developed in response to treatment with various doses
of MTP in MAC, CLTSV.-.S, and B16 tumor-host systems. In
the case of MAC, it was observed that a MTP dose of 5 /¿g
MTP treated
8
9
10
No. of animals
Chart 1. Determination of viable cell inoculum for 50% tumor take for
ClîTSVsS
tumor in untreated and MTP-treated hamsters. Groups of 20
hamsters were each given injections s.c. of different numbers (10*, 103, 104,
105, 10', 107) of CI,TSV.,S cells. One-half of the animals in each group was
treated s.c. with 0.9% NaCI solution, and the other half was treated with 10
ng MTP in 0.9% NaCI solution. The number of animals with tumor ( G) and
without tumor (•)5 weeks after tumor implantation are plotted against the
tumor cell dose. The crossing of the 2 curves (after extrapolation in the case
of MTP-treated animals) gives the viable cell inoculum for 50% tumor take.
MTPfcif iM/mM/ntu
or higher was adequate in preventing tumor appearance in
a maximum number (40%) of the animals and in keeping
the size of the tumor to a minimum, which was 40% smaller
than in the controls. With CLTSV-S tumor, minimum tumor
takes were with a dose of 10 ^g MTP. The number of
animals with tumor, with this dose, was 30% compared to
80% in the controls. The size of tumors that developed was
about the same in animals treated with 0.1 to 50 /ug MTP.
but it was 40 to 50% smaller in MTP-treated animals as
compared to controls. Results with B16 indicated that a
MTP dose of 10 /¿gwas the most effective, giving 20% less
tumor takes as compared to 100% in 0.9% NaCI solutiontreated controls. The size of the tumor was reduced maxi
mally (about 40%) when the MTP dose range was between
1 and 50 ¿¿g.
Action of MTP on the Immune System. On the basis of
the known action of LPS on various arms of the immune
system of the animals, we tested whether MTP (a) was a
mitogen for spleen lymphocytes of Fischer rats, nude mice,
and mice of various strains; (b) was capable of enhancing
serum antibody titers and spleen PFC with SRBC used as
an antigen; and (c) could render in vitro peritoneal macro
phages cytotoxic to tumor cells. The results are shown in
Table 2. It was found that MTP was mitogenic for spleen
lymphocytes of Fischer rats, Swiss mice, AKR mice, and
nude mice. It did not mitogenically
stimulate spleen lym
phocytes of C57BL/J mice and Syrian hamsters. Mitogenic
stimulation of spleen cells was found to peak at 1 to 2 /¿g
MTP per ml in the responding
species and strains of
animals. High concentrations
of MTP (50 jug/ml) inhibited
the background counts of [3H]thymidine incorporated
into
DNA as well as phytohemagglutinin-mediated
stimulation of
spleen lymphocytes (not shown).
MTP treatment of Fischer rats, following i.p. SRBC inoc
ulation, showed a 2- to 4-fold increase in spleen PFC on
Day 4 that depended on the number of SRBC injected
(Table 2). The anti-SRBC serum antibody titers were in
creased 8-fold on Day 7 in.MTP-treated animals (Table 2). In
vitro MTP treatment was also effective in activating perito
neal macrophages of Fischer rats so that they were cyto
toxic for MAC (Table 2).
In a test of whether in vivo MTP treatment rendered
peritoneal macrophage and spleen cells cytotoxic to tumors
cells as well, groups of rats and hamsters were (a) inocu-
3rtini
Chart 2. Relationship of MTP dose and its antitumor response against MAC (A), CI,TSVr,S(6), and B16 (C) tumors The antitumor response was measured
by the number of tumor takes and by the tumor size (length and breadth). The time of observation was 3 weeks after s.c. tumor implantation. The number of
animals that received s.c. 0.9% NaCI solution alone or each of the MTP doses in 0.9% NaCI solution was 10 for each of the Stumors used. Bars, S.D.
3318
CANCER RESEARCH VOL. 38
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Antitumor
Activity
of MTP
Table 2
Immunological reactivities of MTP
Mitogenic stimulation of spleen lymphocytes in
vitro"-b
Spleen lympho
ofSwiss
cytes
Anti-SRBC PFC and antibody responses on
Fischer rats"
PFC response with
macroTreatincorporation(cpm/106cells)1341
sus-ment 5 x 106
1 x 10s
pensionNone SRBC
SRBCrf
Activation of Fischer rat
peritoneal
macropha
ges"'''
ment no.
and addition to
Anti
body titer
phage
inhibi
tion of
[3H]thymidine incor
poration
into
MCA
DNAbymacro
phages19.060.027.280.8
110'3011
±
NoneMTP 10 ±2.8
36.2 ±4.2
128
1.
miceFischer
18 ±3.0 125.0 ±9.6
1024
MTP2.
1.
±312295
None2.
ratsAKR
5321 ±
±2323318
72
None%
miceC57BL/J
5675429
±
±4163834
miceBALB/c,
3573884
±
4851942
±
nudeSyrian
1897766
±
±515573
HamsterAdditionNoneMTPNoneMTPNoneMTPNoneMTPNoneMTPNoneMTP[3H]Thymidine
37363
±
± 32Experi
" For methodology, see "Materials and Methods."
6 The dose of MTP used was 2 /¿g/ml.Dose-response curves with Swiss mice and Fisher rat spleen lymphocytes indicated that a dose
of 1 to 2 /«.g
MTP per ml gave the maximum mitogenic response in the range 0.1 to 50 /j.g MTP per ml tried. The values obtained above are
for 4 Fischer rats and for mixed spleen lymphocytes of 5 mice of each strain in 4 separate experiments.
r The dose of MTP used was 10 /¿g/ml.Dose-response experiments with 0.1, 10, and 50 /ug MTP per ml indicated little response with
0.1 jitg and about 30% lesser response with 50 ^g than that obtained with 10 jug MTP. The values are an average for 2 animals in each
experiment.
ri The PFC dose response with 0.1, 10, and 50 ¿tgMTP injected s.c. per animal gave little response with 0.1 /¿g
and approximately the
same response with 10 and 50 ^g MTP. In these experiments a dose of 10 /¿g
MTP was used. These values were obtained with 4 animals
each in untreated and MTP-treated groups.
e Averages ±S.D.of 5 wells.
lated with their respective tumors alone, (b) treated with
MTP alone, and (c) inoculated with tumor cells and then
treated with MTP. The animals were sacrificed on Day 7 to
test the cytotoxicity of their spleen cells and peritoneal
macrophages against the respective target tumor cells.
MTP treatment alone enhanced the cytotoxicity of perito
neal macrophages but not that of spleen cells. On the other
hand tumor-transplanted animals treated with MTP ex
hibited strong cytotoxicity in their peritoneal macrophages
against the respective target tumor cells. The spleen cells
exhibited slightly higher cytotoxicity as well. Since cultured
rat transformed fibroblasts used as nonspecific target cells
were killed by normal Fischer rat peritoneal macrophages,
we were unable to determine whether MTP action rendered
peritoneal macrophages of MAC-inoculated animals specif
ically cytotoxic to MAC (Table 3). Additional experiments
with several target cells are planned to test the specificity of
cytotoxicity of macrophages derived from MTP-treated,
tumor-inoculated animals.
DISCUSSION
This study clearly demonstrates that a simple "lipid Alike" synthetic compound, MTP, is capable of eliciting
antitumor activity in tumor-transplanted animals. Since
MTP possesses no endotoxic activity and mice tolerate
OCTOBER
1978
without ill effects a dose of MTP 1000-fold greater than the
therapeutic dose, it offers a significant advantage over lipid
A and endotoxin in tumor therapy. MTP can also be injected
repeatedly into animals, whereas repeated endotoxin injec
tions cause animal death (11). During the course of our
work, the papers of Behling ef al. (4) and Rosenstreich ef
al. (14) reported the synthesis of W-fatty acyl D-glucosamines that lacked endotoxic activity but possessed immunopotentiating capabilities. Recently, a paper also ap
peared on the antitumor activity of palmitoyl dextran phos
phate (19).
The antitumor activity of MTP is confined to its ability to
(a) enhance host capacity to reject a larger number of
tumor cells, (b) retard growth in tumor size, and (c) induce
hemorrhagic necrosis in certain tumors. However, 3 other
large fast-growing tumors did not regress under the influ
ence of MTP alone, and an insignificant increase in the life
span of such animals treated with MTP was obtained. Thus,
MTP differed from endotoxin, which has been shown by
Parr ef a/. (11) to cause hemorrhagic necrosis after the
tumor transplant establishes vascularization, and is without
effect when injected on Day 0 after tumor implantation.
However, these authors did not determine whether the
viable cell inoculum for 50% tumor takes of tumor cells was
enhanced on endotoxin treatment starting from Day 0. Our
own unpublished experiments have shown that bacterial
3319
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V. N. Nigam et al.
Table 3
Generation of cytotoxic effector cells by MTP treatment in vivo against MAC and
CI2TSV£
For details of the experiment see "Materials and Methods." The values are averages of
experiments with 4 rats in each group.
% inhibition of [3H]thymidine
incorporation by effector
cells into DNA of
mit.*its
j pt< vi
vivo0.9%
Treatment in
viiwvwi
\->c 11
MACPEC,
ratio to target
±2.5"20.5
100:1PEC,
solution106MACor106CI,TSV,S10MgMTP106MACoM06CI,TSV,SNaCI
3.529.8
±
100:1PEC,
3.181±
100:1PEC,
±4.515.0
.2
100:1Spleen
10MgMTP0.9%
solution10MgMTP10«
NaCI
1.016.0
±
lymphocytes,100:1Spleen
1.515.3
±
±3.218.3
1.848.1
±
3.90.02.5
±
lymphocytes,100:1Spleen
2.129.1
±
1.215.0
±
106CI2TSV5S106MACor106CI2TSV5S+
MAC or
lymphocytes,100:1Spleen
3.544.5
±
1.929.5
±
±3.1
lymphocytes,100:1SpleenMAC16.9 ±2.8CI2TSV5S11.1
10 fig MTPi
" Average±S.D.
glycolipid mR595 is no more effective than is MTP against
growing as well as freshly transplanted MAC, although a
combination of MTP and glycolipid mR595 offers a definite
advantage over MTP treatment alone.
The degree of antitumor activity elicited by MTP depends
on the tumor-host system used. Based on the number of
inoculated tumor cells rejected by the host on MTP treat
ment, MAC was the least sensitive; NH, CI.JSV5S, and B16
were of intermediate sensitivity; and L26 was the most
sensitive. The reason for the different degrees of sensitivity
of various tumors to MTP treatment probably lies with
several factors including immunogenicity of the tumor, the
rate of its growth, and the ability of the tumor to release
factors antichemotactic for macrophages (17).
The mode of antitumor action of MTP appears to be via
its action on the immune system of the host, since the
ability of MTP to reject a small number of tumor cells is
destroyed by 400 rads X-irradiation of animals. By itself
MTP is noncytotoxic to tumor cells in vitro, since viability of
cells as seen by trypan blue exclusion is not affected by
incubation of tumor cells with MTP (5 ¿¿g/ml)
for 2 to 3
days. We also found that MTP mitogenically stimulated
spleen lymphocytes of certain species and strains of ani
mals, although tumor rejection occurred in both mitogenic
responders and nonresponders. Further, MTP enhanced
Fischer rat spleen PFC and serum antibody titers with
SRBC used as antigen. However, antitumor antibody titers
were enhanced only slightly on MTP treatment (Table 2, see
Footnote e). Only animals that had rejected MAC or NH, in
the absence or presence of MTP treatment, showed an
increase in antitumor antibodies. It seems likely that tumor
rejection leads to antibody appearance in the serum rather
than that antibodies cause tumor destruction. However, it
is possible that MTP-induced antitumor antibodies lead to
specific arming of macrophages, cytotoxic to the tumor
cells.
In this study we could also demonstrate that activation of
Fischer rat peritoneal macrophages was obtained by in vitro
3320
MTP treatment. Furthermore, Fischer rats and hamsters
transplanted with their respective tumors and then treated
with MTP gave peritoneal macrophages that were much
more cytotoxic to tumor cells than were those derived from
animals receiving tumor cells of MTP alone. However, we
have no reason to believe that MTP activates macrophages
specifically against the implanted tumor. Additional work
with several tumor target cells is required to show whether
either a greater or a specific activation of peritoneal mac
rophages in tumor-transplanted animals occurs in compar
ison to MTP alone treated animals.
In spite of the presence of activated macrophages in the
MAC-bearing MTP-treated Fischer rats, this tumor is more
resistant to MTP-induced tumor regression than is CI2TSV.-,S
tumor in hamsters. It is possible that either MAC releases
factors that are antichemotactic for macrophages or this
tumor is poorly immunogenic so that T-cells are insuffi
ciently sensitized to deliver a chemotactic signal to acti
vated macrophages.
Although the mode of action of MTP is yet unclear, our
recent comparative experiments (H. El Kappany, C. Chopra,
V. N. Nigam, C. A. Brailovsky, and M. Elhilali, unpublished
results) with BCG and MTP against Dunning R-3327-H
transplantable prostatic carcinoma in Copenhagen-Fischer
F, male hybrid rats demonstrate that MTP was more effec
tive than was BCG when the tumor burden was small. Both
were ineffective against large tumors, even when injected
i.t. However, BCG (13) and BCG cell walls plus trehalose
mycolate (22) have been reported to be very promising
against line 10 hepatocellular carcinoma in guinea pigs.
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3321
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Maltose Tetrapalmitate, a Nontoxic Immunopotentiator with
Antitumor Activity
Vijai N. Nigam, Carlos A. Brailovsky and C. Chopra
Cancer Res 1978;38:3315-3321.
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