[Cancer Research 60, 350-358, January 15, 2000]
© 2000 American Association for Cancer Research
Experimental Therapeutics |
Effective Elimination of Lung Metastases Induced by Tumor Cells Treated with Hydrostatic Pressure and N-Acetyl-L-Cysteine1
Yechiel Goldman2,
Alpha Peled and
Meir Shinitzky3
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
 |
ABSTRACT
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In previous studies, we have demonstrated that application of high
hydrostatic pressure (P) to tumor cells in the presence of a
slow-reacting membrane-impermeable cross-linker (CL), 2'-3'-adenosine
dialdehyde, can rearrange cell surface proteins into immunogenic
clusters. Here, we present evidence indicating that subsequent
reduction of surface protein disulfides with
N-acetyl-L-cysteine (NAC) further augments
the immunogenic potential of PCL-modified tumor cells both in
vitro and in vivo. Immunotherapy with
PCL+NAC-modified 3LL-D122 Lewis lung carcinoma cells plus i.v. delivery
of NAC in mice bearing established lung metastases provoked an
antitumor response capable of eradicating the metastatic nodules as
demonstrated by restoration of normal lung weight and histology. In
addition, immunization with PCL+NAC-modified tumor cells gave rise to a
strong delayed-type hypersensitivity recall response against parental
D122 cells. We propose that this novel two-prong strategy, based on
local immunization with autologous PCL+NAC-modified tumor cells and
systemic boosting with NAC, could provide a practical, effective
immunotherapeutic regimen for the treatment of human cancer.
|
INTRODUCTION
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Immunotherapy of cancer has been of limited clinical value to
date, largely because of its failure to overcome the multiple escape
strategies used by tumor cells (1)
. Renewed interest in
cancer immunotherapy was stimulated in recent years by the
identification of distinct tumor antigens that are capable of eliciting
specific antitumor cytotoxic immune responses in vitro as
well as in vivo (2
, 3) . However, the
availability of synthetic or purified tumor antigens may not in itself
be sufficient to offer an improved therapeutic modality because tumor
antigens are numerous; metastatic tumor cells mutate frequently,
thereby altering their antigenic nature; and the immunogenic hierarchy
of various tumor antigens is largely unknown.
Current strategies to elicit specific antitumor T-cell responses
include gene transfer into autologous tumor cells (4)
,
vaccination with dendritic cells preloaded with tumor antigens
(5
, 6)
, systemic or paracrine cytokine therapy with
Th1-type4
cytokines (i.e., interleukin 2 and granulocyte-macrophage
colony-stimulating factor; Refs. 7
, 8
), vaccination
with T-cell epitopes derived from tumor proteins (2
, 9)
,
and vaccination with tumor-derived heat shock proteins (HSPs; Refs.
10
, 11
). However, tumor cells can evade immune killing
either by down-regulation or total absence of immunogenic surface
molecules, including MHC class I, or by synthesis and secretion of
molecules capable of inactivating cytotoxic effectors, as well as by
defective capillary neovasculature around the malignant tissue that
slows down or even prevents cytotoxic cell egress into the tumor mass
(1)
. Therefore, the strategy of an efficient
immunotherapeutic attack on metastatic cancer must be based on combined
local and systemic approaches capable of coping with such varied escape
mechanisms simultaneously.
Over the last several years, our group has brought hydrostatic pressure
to bear on the augmentation of the immunogenicity of tumor cells. We
have developed a method in which cells are exposed to high hydrostatic
pressure in the presence of a slow reacting biologically compatible
impermeable cross-linker, AdA. This PCL procedure was shown to be
effective and practical for increasing the immunogenicity of both
murine (12, 13, 14, 15, 16)
and human tumor cells (17)
.
Hydrostatic pressure operates by transient depolymerization of
cytoskeletal elements, thus allowing rearrangement of proteins on the
cell surface (18
, 19)
, as well as by induction of putative
immunogenic HSPs (20)
. The net result of these alterations
translates into formation of heterologous protein clusters on the
surface of the treated cells, thereby converting such cells into
potential targets for APCs. Despite being an effective and innocuous
method, PCL by itself still suffers drawbacks similar to those of the
other immunomodulating procedures mentioned above.
Rhodes et al. (21)
observed that carbonyl
groups artificially implanted on the surface of target cells can
interact with free amine groups on the surface of
CD4+ effectors to form stimulatory intercellular
reversible Schiff bases that activate the
Th1-like cytokine secretion pattern. An analogous
intercellular bridge can be induced by infecting malignant cells with
an innocuous strain of the Newcastle disease virus. The viral
antigens that appear on the surface of the infected tumor cells deliver
a specific recognition signal to the responding T cell via
MHC/peptide-TCR interaction. Simultaneously, the Newcastle disease
viral infection introduces a new class of cell-surface adhesion
molecules, hemagglutinin-neuraminidases, which augment tumor
cell-surface adhesiveness for responding effectors (22)
.
The notion that reversible intercellular bridging between target and
effector cells can strengthen specific immunological signals led us to
investigate the effect of the production of reversible intercellular
disulfide bonds in an antitumor immune response. We selected the
biologically compatible reducing agent NAC, a mucolytic agent used in
the treatment of respiratory disorders (23)
, to impose
reactive sulfhydryl groups on the surface of tumor cells. In this
study, we have tested the combination of PCL modification, NAC
sulfhydryl reduction, and i.v. delivery of NAC (24
, 25)
in
the induction of systemic antitumor cellular immunity. As our final
goal, the capacity of this combined regimen to eradicate established
metastasis was investigated.
|
MATERIALS AND METHODS
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Mice
Female C57BL/6 (H-2b) and Balb/C
(H-2d) mice, 8–12 weeks of age, were obtained
from the animal breeding center, Weizmann Institute, Rehovot, Israel.
Animals were maintained and treated according to NIH guidelines.
Tumor Cell Line
The D122 clone of the 3LL Lewis lung carcinoma
(26)
, of C57BL/6 origin, was used. D122 cells were
maintained in RPMI 1640 containing the following supplements, all from
Beit HaEmek, Israel, unless otherwise noted: 10% FCS, glutamine,
combined antibiotics, sodium pyruvate, nonessential amino acids, and 50
µM ß-mercaptoethanol (Merck, Schuchardt, Germany). This
nutrient-rich medium was termed complete medium. Cells were transferred
twice a week and were free of Mycoplasma contamination as
determined by a Mycoplasma ELISA test (Boehringer Mannheim
GmbH, Mannheim, Germany) carried out every 3 months.
Buffers
PB was composed of NaCl solution (saline) containing 8.0 g/l
(150 mM) NaCl (pH 7.4). HBSS was composed of PB plus 1.0
g/l D-glucose (5.0 mM; pH 7.4).
Flow Cytometric Analysis
NAC Titration Assay.
Freshly removed C57BL splenocytes were treated with RBC-lysing solution
(Sigma Chemical Co., St. Louis, MO) and washed twice in PB; aliquots of
3 x 106 cells were incubated with
titrated concentrations of freshly prepared NAC in PB (pH 7.4) for 30
min at 37°C with occasional shaking. After cells were washed twice in
PB, they were incubated with 10 µM F5M or 10
µM F5M-cys, i.e., F5M that was reacted with
cysteine to block its active maleimide function. The blocked probe was
prepared as follows. A solution containing 10
µM F5M and 10 mM
L-cysteine in PB (pH 7.4) was prepared and
reacted at 25°C for 2 h in the dark to form F5M-cys. Conversion
to F5M-cys was assessed by TLC on silica gel 60
F254 (Merck, Darmstadt, Germany) with a running
solvent system of chloroform-methanol-0.1 M
sodium hydroxide (65:25:4, v/v) and was found to be >95% pure. Cells
were incubated with the above probes for 1 h at 4°C, washed, and
immediately analyzed on a FACSort instrument (Becton Dickinson,
Franklin Lakes, NJ).
CD4 and CD8 Analysis.
Rat antimouse monoclonal antibodies were produced by hybridomas GK1.5
(anti CD4) and 53-6 (anti CD8), which were obtained from ATCC, and
mouse antirat-FITC F(ab')2 was obtained from
Jackson ImmunoResearch, West Grove, PA. Freshly removed C57BL
splenocytes were treated with RBC-lysing solution and washed
twice in PB; aliquots of 3 x 106
cells were incubated with anti-CD4 or anti-CD8 antibodies for 30 min at
4°C. After the cells were washed, they were incubated with mouse
antirat-FITC for an additional 30 min at 4°C. Cells were washed and
immediately read on the FACSort.
|
Preparation of AdA
|
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The synthesis of AdA was carried out by reacting adenosine
(Fluka, Buchs, Switzerland) with sodium meta-periodate (BDH,
Poole, United Kingdom) by a modification of the method described
previously (13)
. One volume of a 90 mM
solution of adenosine was reacted with 0.1 volume of a 880
mM solution of sodium meta-periodate by dropwise addition
of the sodium meta-periodate to the adenosine solution while mixing.
The reaction was allowed to proceed for 15 min at room temperature, and
then 0.05 volume of 2 M HCl were added. Quantitative
removal of the iodate byproduct was carried out by the addition of 0.9
volume of a boiling solution of 0.1 M
PbCl2 to the cooled AdA reaction mixture while
stirring to form a precipitate of
Pb(IO3)2. After 15 min on
ice, the precipitate was filtered through three Whatman no. 1 filters
(Whatman, Kent, United Kingdom) under vacuum. The slight excess of
Pb2+ was removed by the addition of 0.02 volume
of 1 M NaH2PO4
and 0.004 volume of 1 M
KH2PO4. A white colloidal
precipitate was formed and was filtered after standing at 4°C
overnight through three Whatman no. 1 filters under vacuum. The
filtrate, composed of 40 mM AdA in an approximate ionic
equivalent of PB, formed the stock solution. The standard working
solution of 10 mM AdA was produced by diluting the stock
solution 1:4 into HBSS. The colorimetric assay of Lappin and Clark
(27)
was used, with minor modifications, to determine the
exact aldehyde concentration of the AdA preparation. The molecular
structure of the AdA was verified by mass spectrometry (data not
shown).
|
PCL
|
|---|
We have optimized the PCL methodology described previously
(16)
. Briefly, a cell pellet containing up to 5 x 107 cells was suspended in 10
mM AdA solution in HBSS, transferred to a sterile
liquipette (Elkay; Precision Laboratory Consumables, Galway, Ireland)
and sealed in an air-free fashion with a sealing clip (Travenol
Laboratories, Teva Medical, Ashdod, Israel). Immediately afterward, the
sealed tube was placed into a custom-designed computer-controlled
pressure device (Advanced Pressure Products, Ithaca, NY). The cells
were then slowly brought to 1200 atm over the course of 15 min, locked
in place for 15 min, and slowly decompressed to atmospheric pressure
again over the course of 15 min such that the rates of compression and
depression were equal. The cells were then washed twice immediately,
resuspended in HBSS, and kept on ice until further use.
|
NAC Modification
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Untreated or PCL-treated cells were suspended in the treatment
buffer, composed of 15 mM NAC (Sigma) and 1% FCS in HBSS
(pH 7.4), and incubated for 30 min at 37°C. Treated cells were
subsequently washed and resuspended in the maintenance buffer, composed
of 15 mM NAC in PB, on ice until further manipulation. In
experiments where the NAC-modified cells were injected into animals,
200 µl of a 0.225 M NAC solution in PB was injected into
the tail veins of mice 
1 h before each inoculation with NAC-modified
cells.
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MLR
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NAC Treatment of Stimulators.
Freshly removed C57BL splenocytes (stimulators) were treated with
RBC-lysing solution, washed twice in PB, and incubated in HBSS alone or
in HBSS containing NAC at 37°C for 30 min. Treated and untreated
stimulators were washed three times and counted. Viability was >95%
in all groups as assessed by trypan blue exclusion. Stimulators were
irradiated with 20 Gy in all MLR assays. Freshly removed Balb/C
splenocytes (responders) from animals previously immunized with
5 x 107 C57BL splenocytes were
isolated as above, washed twice in PB, and counted. Stimulators
(1 x 105 to 1.25 x 104 cells) and responders (1 x 105 cells) were plated into 96-well
microtiter plates in sets of six in a final volume of 200 µl per
well. Plates were incubated for 4 days at 37°C and were pulsed with
[3H]thymidine (6.7 Ci/mmol; ICN, Costa Mesa,
CA) for the last 16 h of the assay. The plates were harvested
(micromate 196 harvester; Packard, Merriden, CT) and scored on a
96-well plate reader (matrix 96; Packard). The stimulation index
(SI) was defined as SI = [(R + S) - R - S]/R, where R
represents the scoring of responding splenocytes alone, and
S represents the scoring of the stimulating splenocytes
alone. A SI value >2 was considered a reliable positive response
(28)
.
Exogenous Addition of NAC to MLR.
Stimulators and responders were prepared as above, and then 20 µl of
150 mM NAC in complete medium (pH 7.4) were added to the
MLR cultures at the beginning of the incubation period. The cultures
were incubated for 3–4 days and harvested as above.
|
MLTR
|
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Ten mice were immunized i.p. four times, 1 week apart, with
2 x 106 PCL+NAC-modified
irradiated (100 Gy) D122 cells. Mice were then challenged with
2 x 105 viable tumor cells s.c.
(100% of these animals remained tumor free); after 4 months, mice were
boosted with 2 x 106
UM-irradiated (100 Gy) D122 cells. One week later, the splenocytes from
these mice were used as responders. Unmodified irradiated (100 Gy) D122
cells served as stimulators. Responders (1 x 105) were cultured with 1 x 105 to 2.5 x 104 stimulators per well in sets of six for 3
days. [3H]Thymidine (6.7 Ci/mmol; ICN) was
added for the last 16 h of the assay. The plates were harvested
(Packard micromate 196 harvester), and radioactivity was scored
on a 96-well plate reader (Packard matrix 96). The SI was calculated as
in the MLR assays.
|
In Vivo Tumor Assays
|
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Survival Assay.
Groups of 9–10 age-matched mice were immunized i.p. four times, 1 week
apart, with 2 x 106 UM or
modified irradiated D122 cells. In all in vivo assays, tumor
cells were irradiated with 100 Gy. Those mice receiving the
NAC-modified cells also received i.v. injections of NAC as described
above. A lethal challenge of 2 x 105 viable tumor cells was introduced s.c. 7 days
after the last immunization, and the percentage of surviving animals
was determined.
Experimental Metastasis Assay.
Groups of 10 age-matched mice were immunized i.p. three times, 1 week
apart, with 2 x 106 UM or
modified irradiated D122 cells, with or without the i.v. NAC, as
described above. One week after the last immunization, 2 x 105 viable D122 cells were injected i.v.
into the tail vein. Three weeks later, the mice were sacrificed, and
the percentage of lung weights above normal (200 mg) was determined.
Metastasis Regression Assay.
Groups of 10 age-matched mice received i.v. injections of 5 x 105 viable D122 cells in the tail vein,
and the cells were allowed to established lung metastases for 8 days
(29)
. On day 9, mice received i.p. injections of
2 x 106 irradiated UM or modified
D122 cells. Those mice receiving the NAC-modified cells also received
NAC i.v. as described above. Two more immunizations, 7 days apart, were
given, and 25 days after the last immunization, the mice were
sacrificed and their lungs were weighed.
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Histological Preparation
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Lungs were removed and immersed in Bouin fixative for 24 h,
washed twice, and resuspended in 70% ethanol. The tissue was mounted
in paraffin blocks, sliced, stained with light green/hematoxylin, and
examined microscopically.
|
DTH Reaction
|
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Groups of five to six age-matched mice received i.p. injections
of 2 x 106 UM ± NAC or PCL ± NAC-modified irradiated D122 cells. Those
mice receiving the UM+NAC or PCL+NAC treatments were boosted 3–4 days
later with a 200-µl injection of 15 mM NAC i.p. This
immunization protocol was repeated twice more, at weekly intervals. One
week after the last immunization, 2 x 105 UM irradiated D122 cells in 50 µl of HBSS
were injected into the right ear and 50 µl of HBSS into the left ear
of each mouse. After 24 h, mice received i.p. injections of a
200-µl solution of 125 µg/ml (1 mM)
5-fluoro-2'-deoxyuridine (Sigma); 30 min later, they received i.p.
injections of 2 µCi of 125I-Urd (2000 Ci/mmol;
Amersham, Buckinghamshire, United Kingdom; Refs. 30
, 31
).
After another 24 h, mice were sacrificed and the ears were
removed. The radioactivity of each ear was then counted in a gamma
counter (Riastar; Packard) and the R/L ratio determined. The upper
limit for the R/L ratio was taken as 2, which is the maximal response
obtained against sheep RBCs in the original assay (30)
.
|
Statistical Analyses
|
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Two-tailed P values were determined using either the
nonparametric Wilcoxon test or Student’s t test to
interpret the significance of the difference between the experimental
groups.
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RESULTS
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Increased Immunogenicity of NAC-treated Cells.
Fresh C57BL splenocytes were incubated with increasing concentrations
of NAC in PB for 30 min at 37°C and then labeled with the fluorescent
probe F5M (see "Materials and Methods"). As a control, F5M was
first reacted with L-cysteine and then used to estimate the
nonspecific binding. The FACS analysis indicated that after treatment
with 10 mM NAC, only a moderate-staining population was
produced with an average mean fluorescence channel of 180 units. In
contrast, at 15 mM NAC, there was a significant shift in
the mean fluorescence channel (average of 790 units), with 14% of
the population staining heavily, thereby demonstrating the ability of
NAC at this concentration to efficiently expose surface thiols (Fig. 1
). Viability was unaffected by treatment with up to 15 mM
NAC (>95%) as determined by trypan blue exclusion.
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Fig. 1. Exposure of cell surface thiols after NAC treatment. C57BL
splenocytes (3 x 106) were incubated with
different concentrations (1, 5, 10, and 15 mM) of
freshly prepared NAC in PB (pH 7.4) for 30 min at 37°C. After
washing, surface sulfhydryl residues were reacted with 10
µM F5M or with 10 µM F5M-cys, which served
as a control of nonspecific binding (see "Materials and Methods").
Treated and untreated cells (UM) were incubated
with the above probes for 1 h at 4°C, washed, and immediately
analyzed by FACS. These results are representative of three repeated
experiments. The background nonspecific binding did not exceed 5 units
per sample (data not shown).
|
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The immunological effect of exposure of surface thiol groups on
splenocytes was assessed using a MLR in which splenocytes of immunized
female Balb/C (H-2d) mice acted as responders (R)
and UM or NAC-reduced, irradiated splenocytes of female C57BL6/J
(H-2b) mice, acted as stimulators (S). The SI of
the allogeneic responders (R/S ratio of 1:1, see legend of Fig. 2
) increased in a dose-dependent manner with increasing
concentrations of NAC in the treatment buffer of the stimulators and
reached a maximum of 21.7 SI units at 10 mM NAC (Fig. 2A
). Similar results were obtained by increasing the
concentration of exogenous NAC added to the medium at the beginning of
the MLR assay, with a maximal value of 33.7 SI units being observed
again at 10 mM NAC (Fig. 2B
). In
parallel, the control syngeneic MLR was performed in which responders
from a Balb/C mouse immunized with Balb/C splenocytes were incubated
with treated syngeneic stimulators under the same conditions of the
allogeneic MLR. As shown in Fig. 2C
, syngeneic responders
did not proliferate in the presence of untreated targets, NAC-reduced
targets, or when NAC was added exogenously to the MLR cultures (all SI
values <1). Given the fact that the reducing capacity of 15
mM NAC was significantly >10
mM and that the immunostimulatory capacity was
similar to that of 10 mM, we chose 15
mM as the standard NAC concentration in
subsequent experiments.
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Fig. 2. A, enhancement of the proliferative
response in allogeneic MLR induced by NAC treatment. Splenocytes
[1 x 105; responders (R)]
from a Balb/C (H-2d) mouse preimmunized with C57BL6
(H-2b) splenocytes were cultured with 1 x 105 (R/S 1:1) to 1.25 x 104 (R/S 1:0.125) UM and NAC-modified C57BL6
(H-2b) splenocytes [stimulators (S)] for 4
days. [3H]Thymidine (1 µCi) was added for the last
16 h of the assay, and the plates were scored for
[3H]thymidine uptake. B, exogenous
addition of NAC to MLR cultures stimulated the proliferative response
of allogeneic splenocytes. Splenocytes (1 x 105) from a Balb/C mouse (R) preimmunized with C57BL6
splenocytes were cultured with 1 x 105
C57BL6 splenocytes (S) in the presence of increasing concentrations of
NAC for 3 or 4 days. [3H]Thymidine (1 µCi) was added
for the last 16 h of each assay, and the plates were scored for
[3H]thymidine uptake. C, in contrast,
NAC-treated syngeneic stimulators (NAC-treat.) as well
as NAC added exogenously to syngeneic MLR culture medium
(NAC-exog.) did not stimulate a proliferative response.
Here, Balb/C mice were immunized with Balb/C splenocytes, and the
splenocytes from the immunized mice were used as responders against
syngeneic targets, with the same R:S ratios as in A. The
results are presented in SI units (see "Materials and Methods") and
are representative of three repeated experiments for each set.
|
|
The in vivo immunostimulatory capacity of NAC was tested as
follows. Ten C57BL6 mice were immunized with 5 x 107 Balb/C splenocytes. Eight days later, after
the establishment of an active primary immune response, five mice
received i.v. injections of NAC (367 mg/kg; final serum concentration,
15 mM), whereas the remaining five mice
received i.v. injections of PB; the splenic CD4 and CD8 expression was
then analyzed by FACS. A significant increase in the relative
expression of CD4 from 8 to 16% with no significant change in the
expression of CD8 was observed in the allogeneic setting (Fig. 3
). In a parallel experiment with naive mice, there was no difference in
the expression of either CD4 or CD8 between the PB and NAC i.v groups
(data not shown).
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Fig. 3. Administration of NAC i.v. stimulates the production of
CD4+ T cells in the spleens of mice immunized with
allogeneic splenocytes. Ten C57BL6 mice were immunized with
5 x 107 Balb/C splenocytes i.p. Eight
days later, five mice received PB i.v. and five mice received standard
NAC i.v., and the splenic expression of the CD4 and CD8 markers was
followed for 5 days post i.v. by FACS analysis. The above results were
observed on day 3 post PB/NAC i.v. and are representative of three
repeated experiments. PBS, phosphate buffer.
|
|
The immunotherapeutic potency of the NAC modality was tested with the
nonimmunogenic D122 clone of the Lewis lung carcinoma, 3LL
(32)
. Accordingly, an NAC treatment protocol composed of
the following three stages was designed: (a) incubation of
the cells in HBSS containing 15 mM NAC and 1%
FCS (pH 7.4) at 37°C for 30 min; (b) washing and injection
of the NAC-modified cells in PB containing 15 mM
NAC (pH 7.4); (c) i.v. injection of NAC to achieve a final
serum concentration of 15 mM (367 mg/kg) 1
h before each inoculation with NAC-treated tumor cells. In a survival
assay, mice were inoculated i.p. four times, 1 week apart, with
2 x 106 irradiated UM or
NAC-modified D122. One week after the last immunization, mice were
challenged with 2 x 105 viable
D122 cells s.c. The percentage of tumor-free animals in the treatment
group was more than twice that of the group treated with UM cells (89%
versus 42%; Fig. 4
).
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Fig. 4. Induction of protective immunity with NAC-modified
3LL(D122) cells. Groups of C57BL6 mice received four i.p. injections, 7
days apart, of either HBSS or 2 x 106
irradiated UM or NAC-modified D122 cells. Those mice receiving the
NAC-modified cells were also administered NAC i.v. as described in
"Materials and Methods." Each group contained 9–10 age-matched
mice. Seven days after the last inoculation, mice were challenged with
2 x 105 D122 cells s.c., and the survival
was followed for 80 days post challenge.
|
|
Following up on the survival experiment, we used the NAC protocol in an
experimental metastasis assay. Here, mice were inoculated i.p. three
times, 1 week apart, with 2 x 106
irradiated UM or NAC-modified cells, and 1 week after the last
injection, 2 x 105 viable D122
cells were administered i.v. into the tail vein. Twenty-five days
later, mice were sacrificed, and their lungs were weighed and scored
for metastatic load with respect to the average normal lung weight (200
mg). As presented in Fig. 5
, there was only a slight difference between the UM-NACiv (80% above
200-mg level) versus the UM+NACiv (70% above 200-mg level)
groups in lung weights. However, there was a significant difference
between the NACmod-NACiv (70%) versus the NACmod+NACiv
groups (25%). These experiments indicated that the combination of
local immunization with NAC-treated tumor cells in addition to systemic
NAC administration improved the antimetastatic activity considerably.
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Fig. 5. Assessment of lung metastasis in mice bearing lung tumors
after various prophylactic immunizations. Groups of 9–10 C57BL6 mice
were immunized i.p. three times, 1 week apart, with 2 x 106 irradiated UM or NAC-modified D122 cells with or
without i.v. administration of NAC (see "Materials and Methods").
Seven days after the last immunization, mice were challenged with
2 x 105 viable D122 cells injected i.v.
After 3 weeks, the mice were sacrificed and the lung weights were
determined. The percentage of lungs above the upper normal lung weight
(200 mg) is depicted (left). NACmod+NACiv group compared
with other groups: versus HBSS group,
P = 0.0007; versus
UM-NACiv, P = 0.0085;
versus UM+NACiv, P = 0.09;
versus NACmod-NACiv, P = 0.07. Photographs of three representative lungs for each group are also
presented (right).
|
|
Combination of PCL and NAC Modifications in Antitumor Immune
Response.
The capacity of splenocytes from mice immunized with PCL+NAC-modified
D122 cells to proliferate in the presence of UM parental tumor cells
in vitro was tested. Splenocytes from PCL+NAC-immunized mice
proliferated (maximum SI, 5.4) in a dose-dependent manner against
decreasing numbers of UM D122 targets in a MLTR (see "Materials and
Methods") as illustrated in Fig. 6
. Splenocytes from naive mice did not proliferate in the presence of the
tumor cells (SI <1; data not shown).
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Fig. 6. Responding splenocytes from PCL+NAC-immunized mice
proliferate in the presence of their unmodified syngeneic tumor targets
(stimulators) in a MLTR. C57BL6 mice were immunized i.p. four times, 7
days apart, with 2 x 106 irradiated
PCL+NAC-modified D122 cells. One week after the last immunization, mice
were challenged with 2 x 105 D122 cells
s.c.; after 4 months, tumor-free mice were boosted with 2 x 106 irradiated UM D122 cells. Splenocytes from
these mice were used as responders in the MLTR, whereas UM-irradiated
D122 cells served as stimulators. Responders (1 x 105) were cultured with 1 x 105
to 2.5 x 104 stimulators for 3 days.
[3H]Thymidine (1 µCi) was added for the last 16 h
of the assay, and the plates were then scored for
[3H]thymidine uptake. The results are presented as SI
units. Values >2 were considered significant (see "Materials and
Methods").
|
|
The efficacy of the combined PCL and NAC modifications in evoking
regression of established metastases was then tested in
vivo. Animals first received i.v. injections of 5 x 105 D122 cells, which were allowed to
settle and to develop metastatic foci in the lungs over the course of 8
days. On day 9, immunotherapy was instituted by inoculation with
2 x 106 irradiated UM or
PCL-modified D122 cells i.p., with or without NAC modification
(including NAC i.v.). Overall, three inoculations were given 7 days
apart, for three consecutive weeks. Mice were sacrificed 25 days after
the last immunization, and the lungs were weighed. The results are
presented in Fig. 7
. As shown, in contrast to the mean lung weight of 420 ± 100 mg (mean ± SE) in mice treated with UM cells only,
mice treated with PCL+NAC-modified cells demonstrated a normal lung
weight of 200 ± 18 mg (Fig. 7A
). Examination
of the histological sections of the lungs correlated with the
metastatic load in each treatment group and clearly indicated the
eradication of the metastatic nodules and recuperation of the alveolar
spaces in the mice inoculated with PCL+NAC-treated cells (Fig. 7B
).
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Fig. 7. A, PCL synergizes with NAC to eradicate
established lung metastases in a metastasis regression model. Groups of
10 C57BL6 mice received i.v. injections of 5 x 105 viable D122 cells. Nine days later, when lung
metastases were well established, mice received i.p. injections of
HBSS, 2 x 106 irradiated unmodified
(UM), or PCL-modified D122 cells with or without NAC
treatment. Two more identical immunizations, each 7 days apart, were
administered. Twenty-five days after the last immunization, the mice
were sacrificed and the lung weights determined. The horizontal
line indicates the upper level of normal lung weight (200 mg).
The results are presented as mean weight; bars, SE.
B, photomicrographs of histological sections from
representative lungs of the mice immunized with HBSS, as well as UM,
UM+NAC-, PCL-, and PCL+NAC-modified D122 cells.
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The efficacy of the antitumor T-cell response in vivo
demonstrated in Fig. 7
was quantified by the radiometric DTH test of
Vadas et al. (30)
. Mice were immunized three
times, 1 week apart, with 2 x 106
UM ± NAC or PCL ± NAC-modified
irradiated D122 cells i.p.; those receiving UM+NAC- or PCL+NAC-modified
cells were boosted between immunizations with NAC i.p. The higher ratio
of the 125I-Urd uptake by the responding
lymphocytes in the right ear (parental tumor challenge)
versus the left ear (buffer) in the PCL+NAC immunization
group lent further support to the contention that immunization with
PCL+NAC-modified tumor cells triggered a specific T-cell immune
response that can be recalled in a DTH assay (Fig. 8
).
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Fig. 8. Immunization with PCL+NAC-modified D122 cells stimulates a
DTH reaction against UM D122 targets. During week 1, groups of 5–6
mice received i.p. injections of 2 x 106
UM, UM+NAC, PCL, or PCL+NAC-irradiated D122 cells; those mice receiving
the UM+NAC and PCL+NAC treatment were then boosted 3–4 days later with
a 200-µl injection of 15 mM NAC i.p. This immunization
protocol with NAC boosting in the UM+NAC and PCL+NAC groups was
repeated twice more, during weeks 2 and 3. Mice immunized with UM or
PCL cells alone were administered PB at the time of NAC boosting. A
quantitative DTH test was performed where UM D122 cells were injected
into the right ear (R) and buffer into the left ear
(L). The R/L ear uptake (mean ± SE) of
125I-Urd, a quantitative measure of the antitumor T-cell
response, is presented. PCL+NAC immunization compared with other
groups: versus HBSS, P = 0.0081; versus UM, P = 0.0255; versus UM+NAC, P = 0.0866; versus PCL, P = 0.0475.
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DISCUSSION
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Successful application of immunotherapy as a modality in cancer
treatment must be able to trigger an effective immune response against
several tumor antigens simultaneously. Subjecting tumor cells to
hydrostatic pressure in the presence of a biologically compatible
cross-linker provides a simple, effective means for augmenting the
expression of a battery of immunologically relevant surface molecules
(16
, 17) . In this study, we have used a two-pronged
strategy composed of immunization with PCL+NAC-modified cells and
systemic administration of NAC as a general T-cell immunostimulant
(24)
. The most notable result was obtained in a metastatic
regression model in which mice received i.v. injections of highly
metastatic D122 tumor cells and lung micrometastases were allowed to
develop over an 8-day period (29)
. Subsequently, a series
of immunotherapeutic regimens were administered, commencing on day 9.
As shown in Fig. 7A
, there is a clear trend of reduction in
mean lung weight progressing from the UM to the PCL+NAC immunization
groups. This finding is supported by the histological findings in Fig. 7B
, which confirmed that the surface modification of the
tumor cells by PCL+NAC in the presence of systemic immunostimulation
with NAC induced a potent antitumor immune response capable of
eradicating established metastases. Although the P value of
PCL+NAC group versus PCL group in the regression assay was
not statistically significant, the P value between PCL+NAC
versus PCL inoculation groups in the DTH test was
significant (P = 0.0475). Taken together, the
emerging picture from Figs. 7
and 8
clearly indicates that the PCL+NAC
regimen implemented in this study is superior to either PCL or NAC
alone. The antitumor activity of this novel innocuous methodology
compares well with other experimental immunotherapeutic approaches
(33, 34, 35, 36)
.
In our previous work, immunization with PCL-modified B16.BL6 melanoma
cells was shown to be effective in protecting against a subsequent
lethal s.c. challenge of B16 cells (14)
and in inducing a
clear DTH recall response against EL4 and ARadLV 136 leukemia cells
(13)
. However, at that point in the development of the method,
immunization with PCL-modified cells was only partially effective in
protecting against a lethal systemic challenge. The primary feature of
the PCL+NAC methodology is its ability to lessen the metastatic load to
an undetectable level. Further support for the potency of the PCL+NAC
treatment emerged from the in vitro MLTR results where
splenocytes from PCL+NAC-immunized mice responded strongly when
cultured in the presence of UM D122 cells (Fig. 6
).
The importance of thiols, (e.g., ß-mercaptoethanol) in
enhancing the growth of cells in tissue culture has been known for >20
years (37)
, but the exact mechanism has been a matter of
long-standing debate (38)
. A number of studies have
further shown that the presence of free thiols in the tissue culture
medium is essential for lymphocyte proliferation and activation
(24
, 39, 40, 41)
. On the basis of findings that NAC can
substitute for cysteine in tissue culture (42)
, we have
tested in this study the ability of NAC as a costimulator in various
in vitro assays. Our working hypothesis states that in the
presence of NAC, surface intracellular disulfide bonds on the effector
and/or target cell will be reduced to free sulfhydryls, thereby
promoting the formation of transient intercellular disulfide bridges
that could initiate a cascade of signal transduction inside the
effector T cell, culminating in activation. In a parallel model,
modification of target cells with carbonyl groups led to the formation
of transient intercellular Schiff bases between the target carbonyl
groups and amines constitutively expressed on presenting cell and
T-cell surfaces, thus providing a strong costimulatory signal to CD4
Th-cells (21)
.
In view of the above, NAC was chosen as an innocuous reducing agent for
exposing surface thiols groups on splenocytes as well as on tumor cells
(Fig. 1
). In the allogeneic MLR assay, NAC-treated targets, as well as
NAC itself added exogenously to the culture medium, were able to
significantly augment the proliferation of primed allogeneic effectors
(Fig. 2, A and B
) but had no effect in the
parallel syngeneic MLR (Fig. 2C
). Furthermore, in a survival
assay, NAC-modified D122 cells induced protection from a lethal
challenge of parental D122 (Fig. 4
).
As to the mechanism of the effector stimulation in the presence of
thiol-enriched target surface, we showed that i.v. administration of
NAC doubled the level of CD4+ splenocytes in mice immunized with
allogeneic splenocytes, whereas the level of CD8+ cells remained
unchanged 3 days after i.v. administration of NAC (Fig. 3
). The
origin of this effect may be in an increase of the half-life of the
cell-APC coupling, which could increase the rate of transfer of an
immunological signal. Lanzavecchia et al. (43)
recently reviewed the kinetics of TCR activation. They pointed out that
MHC/peptide complexes do not need to bind with high affinity to trigger
the TCR. Strong TCR agonists are typically characterized by a
Kd of 1–90 µM
and half-lives of 10 s (44)
. In line with these values,
it is suggested that the reactive thiols on the surface of target cells
form transient intercellular bridges, which prolong the time of
interaction between the effector and the stimulator and/or APC, thereby
enhancing the rate of transfer of the specific immunological signal.
This proposed mechanism could add another dimension to the kinetic
proofreading model (45)
by furnishing additional
activation signals to T lymphocytes that could aid in breaking the
nonresponsiveness of the immune system to disseminated metastatic foci.
Our results demonstrated that i.v. administration of NAC in addition to
the NAC-modified tumor cells contributes significantly to the antitumor
response. Taking into account the estimated 2-h mean half-life of the
reduced form of NAC in the serum (23)
, we injected mice
with a small volume of a concentrated NAC solution to reach a systemic
concentration of reduced NAC of 15 mM in the serum 1
h before the NAC-treated tumor cells were injected. The rationale
behind this was to present the host immune system with a specific
immunogenic target and a systemic T-cell stimulant simultaneously. This
combination synergized in the experimental metastasis model, where NAC
surface modifications plus NAC i.v. (NACmod + NACiv group)
proved to be the most effective in reducing the metastatic load, as
seen both in the small (25%) percentage of lung weights above the
normal 200-mg lung weight, as well as the gross appearance of the lungs
themselves (Fig. 5
).
In a preliminary set of
experiments,5
we detected an increase in the surface expression of HSP90 and HSP96 on
the surface of PCL-treated B16.BL6 murine melanoma cells. Strong
evidence is presented in the literature that these HSPs can serve as
collectors and transporters of immunological peptides from the
cytoplasm to the exterior plasma membrane. Indeed, vaccination with
HSP70, HSP90, and GP96 was shown to elicit specific immunity against
the tumors from which they were isolated (10
, 46)
. On the
basis of these converging lines of evidence, we hypothesize that
PCL+NAC cells in the inoculum present immunogenic HSPs in addition to
other immunogenic components on their surfaces and possibly leak
HSP-peptide complexes into their immediate vicinity. APCs responding to
these "danger signals" (47)
pick up these
immunological complexes, process them, and present them to both CD8+
and CD4+ T cells. Circulating NAC stimulates these T cells in a
positive feedback loop, thereby augmenting their capacity to respond to
the remaining metastatic cells. Further clarification of the exact
mechanism underlying the immunogenic potency of the PCL+NAC-treated
cells is currently under investigation.
The antitumor potency of the combined PCL+NAC protocol stands out in
its efficacy and applicability to the clinical setting. The advantage
of this protocol manifested itself in the quantitative measurement of
DTH to UM D122 cells after immunization with PCL+NAC-modified cells
(Fig. 8
). This immunization was sufficient to produce a significant DTH
recall response to parental D122 cells in vivo. It is
thus apparent that the PCL+NAC modification, combined with the NAC i.v.
administration, is capable of breaking the barrier of nonresponsiveness
of the immune system to the nonimmunogenic D122 tumor cells. The
regimen of local immunization with PCL+NAC-modified tumor cells and
systemic NAC administration meets the stringent criteria of a
"multivalent" tumor vaccine that may eventually be applied in the
clinic.
ACKNOWLEDGMENTS
We thank Drs. Avi Eisenthal, Amnon Gonnene, Cohava Gelber, and
David Dove for fruitful discussions, and Rachel Haimovitz for technical
assistance.
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FOOTNOTES
|
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was partially supported by a grant
from Immunotherapy Inc., New York. 
2 The studies of Yechiel Goldman have been
supported in part by the Dr. Judith Schneider and Dr. E. Richard
Feinberg Scholarship.
3 To whom requests for reprints should be
addressed, at the Department of Biological Chemistry, Weizmann
Institute of Science, Rehovot 76100, Israel. Phone: 972-8-934-2750;
Fax: 972-8-934-4112; E-mail: bmshinit{at}weizmann.weizmann.ac.il
4 The abbreviations used are: Th1,
type 1 helper phenotype; HSP, heat shock protein; AdA, adenosine
dialdehyde; PCL, pressure cross-linking; APC, antigen-presenting cell;
TCR, T-cell receptor; NAC,
N-acetyl-L-cytsteine; PB, phosphate buffer;
F5M, fluorescein-5-maleimide; F5M-cys, F5M-cysteine-blocked probe;
MLR, mixed lymphocyte reaction; MLTR, mixed lymphocyte-tumor reaction;
SI, stimulation index; UM, unmodified; 125I-Urd,
[125I]-iodo-2'-deoxyuridine; DTH, delayed-type
hypersensitivity; R/L ratio, right/left ratio; FACS,
fluorescence-activated cell sorting; NACiv, NAC i.v.; NACmod,
NAC-modified.
5 Y. Goldman et al.
Pressure-induced heat shock proteins promote the immunogenicity of
tumor cells, manuscript in preparation.
Received 8/ 2/99.
Accepted 11/11/99.
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ABSTRACT
INTRODUCTION
