Received: January 13, 2016; Revised: July 11, 2016; Accepted: September 12, 2016
© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Carcinogenesis, 2016, Vol. 37, No. 12, 1170–1179
doi:10.1093/carcin/bgw099
Advance Access publication September 22, 2016
Original Manuscript
1170
The triterpenoid CDDO-imidazolide reduces immune cell
inltration and cytokine secretion in the Kras
G12D
;Pdx1-
Cre (KC) mouse model of pancreatic cancer
Ana S.Leal
1,2
, Michael B.Sporn
1
, Patricia A.Pioli
3
and Karen T.Liby
1,2,
*
1
Department of Pharmacology, Geisel School of Medicine at Dartmouth, Hanover, NH 03756, USA,
2
Department of
Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824, USA and
3
Department of Microbiology and
Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA
*
To whom correspondence should be addressed. Department of Pharmacology and Toxicology, Michigan State University, B430 Life Science Building, 1355
Bogue Street, East Lansing, MI 48824, USA. Tel:+1 517 884 8955; Fax: +1 517 353 8915; Email:
liby.kare@msu.edu
Abstract
Because the 5-year survival rate for pancreatic cancer remains under 10%, new drugs are needed for the prevention and
treatment of this devastating disease. Patients with chronic pancreatitis have a 12-fold higher risk of developing pancreatic
cancer. LSL-Kras
G12D/+
;Pdx-1-Cre (KC) mice replicate the genetics, symptoms and histopathology found in human pancreatic
cancer. Immune cells inltrate into the pancreas of these mice and produce inammatory cytokines that promote tumor
growth. KC mice are particularly sensitive to the effects of lipopolysaccharide (LPS), as only 48% of KC mice survived an
LPS challenge while 100% of wildtype (WT) mice survived. LPS also increased the percentage of CD45+ immune cells in
the pancreas and immunosuppressive Gr1+ myeloid-derived suppressor cell in the spleen of these mice. The triterpenoid
CDDO-imidazolide (CDDO-Im) not only reduced the lethal effects of LPS (71% survival) but also decreased the inltration
of CD45+ cells into the pancreas and the percentage of Gr1+ myeloid-derived suppressor cell in the spleen of KC mice 4–8
weeks after the initial LPS challenge. While the levels of inammatory cytokine levels were markedly higher in KC mice
versus WT mice challenged with LPS, CDDO-Im signicantly decreased the production of IL-6, CCL-2, vascular endothelial
growth factor and G-CSF in the KC mice. All of these cytokines are prognostic markers in pancreatic cancer or play
important roles in the progression of this disease. Disrupting the inammatory process with drugs such as CDDO-Im might
be useful for preventing pancreatic cancer, especially in high-risk populations.
Introduction
Over 48 000 new cases of pancreatic cancer will be diagnosed
in the USA in 2015, and this disease is almost uniformly fatal,
with 5-year survival rates still below 10% (1). Pancreatic ductal
adenocarcinoma (PDAC) is resistant to conventional cytotoxic
chemotherapy and radiotherapy, and more than 80% of patients
present with unresectable or metastatic disease. Gemcitabine
has been the standard palliative therapy for patients with
pancreatic cancer for more than a decade (2). In a phase III
clinical trial in patients with advanced pancreatic cancer, a
combination chemotherapy regimen composed of FOLinic
acid, 5-Fluorouracil, IRINotecan and OXaliplatin (FOLFIRINOX)
improved survival 4months longer than gemcitabine (11.1 ver-
sus 6.8months (3)), but this drug regimen is too toxic for most
patients. In 2013, the FDA approved nab-paclitaxel (Abraxane)
as a less toxic alternative (4), but this new drug is less effective
than FOLFIRINOX, and none of these newer therapies extend
survival beyond a year. Although prevention may become the
most effective strategy for reducing the unacceptable mortal-
ity rates for pancreatic cancer, new drugs and approaches are
needed for this devastating disease.
The development of relevant genetically engineered mouse
models has provided important biological insights into the
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A.S.Leal et al. | 1171
pathogenesis of PDAC (5). Activating mutations in the Kras
gene are found in over 90% of human pancreatic cancers. The
introduction of a Kras mutation specically in the pancreas
of KC mice (genotype = LSL-Kras
G12D/+
;Pdx-1-Cre) closely repli-
cates the genetic background, clinical symptoms and full spec-
trum of histopathology found in human PDAC (6). Asignicant
inammatory response is also characteristic of human PDAC
(7). Inltrating immune cells can account for up to 50% of the
cells in a pancreatic tumor, and both the immune cells and the
tumor produce cytokines and chemokines that suppress anti-
tumor immunity and promote tumor development (8). Notably,
early inltration of immune cells, including tumor-associated
macrophages (TAMs), myeloid-derived suppressor cells (MDSCs)
and regulatory T cells (Tregs), has also been observed in KC mice
(9). These cells drive tumor progression, so targeting these cells
early in the disease process might be useful for altering the
pathogenesis of this disease.
Although activation of Kras during embryonic development
is sufcient to induce PDAC, acute and chronic pancreatitis
accelerates the development of pancreatic cancer in mice with
Kras mutations (10–12). Epidemiologic studies have found that
patients with chronic pancreatitis have a 12-fold higher risk
of developing PDAC (13). Caerulein, a CCK analog, is one of the
most widely used inducers of pancreatic injury in animal mod-
els. Despite being a useful reagent for inducing acute pancrea-
titis, caerulein must be injected almost daily for several months
(10) to induce chronic pancreatitis. In contrast, when injected
i.p. once a week for 4 weeks (11), lipopolysaccharide (LPS)
induces chronic inammation and early pancreatic lesions in
mice with an activating Kras mutation in pancreatic acinar cells.
LPS is not routinely used to induce pancreatitis (14) in animal
models, even though it is widely used to trigger an inamma-
tory response, including the release of inammatory cytokines
from macrophages, both in vitro and in vivo. The effects of LPS-
induced inammation on immune cell inltration and cytokine
secretion in KC mice are notknown.
Because of the importance of inammation and immune
cell inltration in carcinogenesis, anti-inammatory drugs
might be useful interventions (15) for pancreatic cancer.
Triterpenoids such as oleanolic acid, found in a variety of plants,
have weak anti-inammatory and anticarcinogenic properties
(16). 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid-methyl
ester (CDDO-Me) and CDDO-imidazolide (CDDO-Im) are syn-
thetic derivatives of oleanolic acid that are 10 000 times more
potent at suppressing production of the inammatory mediator
inducible nitric oxide synthase than the parent oleanolic acid
molecule. Synthetic triterpenoids also inhibit the production of
IL-1β, IL-6, TNFα, vascular endothelial growth factor (VEGF) and
CCL2 in both immune and cancer cells (
16). In mice challenged
with a low dose of LPS, CDDO-Me suppresses the production
of proinammatory cytokines and alters the composition of
immune cell populations in the spleen; CDDO-Me also reduces
the number of deaths in mice injected with lethal doses of LPS
(
17). Moreover, CDDO-Me suppresses angiogenesis in vivo (18),
reduces the inltration of TAMs into ER-mammary tumors in
PyMT mice (19), reprograms TAMs from an M2 tumor promot-
ing phenotype to an M1 tumor inhibiting phenotype (20) and
inhibits the immunosuppressive activity of MDSCs in mice and
in cancer patients (21). CDDO-Me is currently being tested in a
Phase II clinical trial for the amelioration of pulmonary arterial
hypertension; RTA408 (22), a second generation triterpenoid, is
being evaluated for its ability to reprogram the immune system
and attack tumors by inhibiting MDSC activation and activating
cytotoxic T cells. We have previously shown that triterpenoids
extend survival in the KPC mouse model of pancreatic cancer
but did not study their effects on the tumor microenvironment
in this model (23). In addition to characterizing the effects of
LPS in KC mice, we tested the ability of CDDO-Im to decrease
cytokine production and immune cell inltration in KC mice.
Materials and methods
Mousemodels
All animal studies were done in accordance with protocols approved by
the Institutional Animal Care and Use Committee at Dartmouth Medical
School and Michigan State University. In KC (LSL-Kras
G12D/+
;Pdx-1-Cre)
mice, mutant Kras is expressed in the pancreas (
6). These mice were gen-
erated by breeding male LSL-Kras
G12D/+
;Pdx-1-Cre and female Pdx-1-Cre
mice. For genotyping, the Extract-N-Amp tissue PCR kit (Sigma) was used
to extract genomic DNA from tail snips (
23). At 4 weeks of age, all KC mice
were randomized and fed powdered 5002 rodent chow (LabDiet) for the
duration of the studies.
For the initial (
Table1) LPS pilot studies, 9-week-old KC mice, WT LSL-
Kras
G12D/+
or Pdx-1-Cre mice or polyoma-middle T (PyMT) mice on the same
C57/BL6 background were injected i.p. with saline (vehicle) or LPS (4 mg/kg,
Sigma O111:B4 lot 084M4118V) once a week for 4 weeks. In protocol 1
(
Figure1), the pancreas and spleen of KC mice were harvested 4 or 8 weeks
after the initial LPS injection (13 or 17 weeks of age). The percentage of
CD45+ immune cells in the pancreas and the percentage of GR1+ MDSC in
the spleen were analyzed by ow cytometry and by immunohistochemistry
(IHC). Aportion of the pancreas from these initial LPS studies was also sec-
tioned and stained with hematoxylin and eosin (H & E). In a pilot study of
ve-paired mice per group harvested 12 weeks after the initial LPS injection,
the number of PanINs and areas of PDAC from 4 to 8 sections per mouse
were counted. Basal differences in CD45+ and Gr1+ MDSC cells in WT and KC
mice were also analyzed by ow cytometry (
Supplementary Figure1, avail-
able at Carcinogenesis Online). To study acute cytokine production (protocol
2,
Figure 2 and Supplementary Tables 1 and 2, available at Carcinogenesis
Online), 9-week-old KC mice were fed CDDO-Im (
24,25) mixed into powdered
LabDiet 5002 chow (100 mg/kg diet or ~25 mg/kg body weight) or control 5002
chow. These diets were started 2days prior to being challenged with a single
dose of LPS (4 mg/kg) or saline (vehicle). Twenty-four hours after the injec-
tions, plasma and pancreas were harvested and analyzed by a multiplex
Abbreviations
CDDO-Im CDDO-imidazolide
CDDO-Me CDDO-methyl ester
ELISA enzyme-linked immunosorbent assay
IHC immunohistochemistry
LPS lipopolysaccharide
MDSC myeloid-derived suppressor cell
OA oleanolic acid
PDAC pancreatic ductal adenocarcinoma
TAM tumor-associated macrophage
Tregs regulatory T cell
VEGF vascular endothelial growth factor
WT wildtype
Table1. KC pancreatic cancer mice are susceptible to LPS
Mouse strain Survivors
P value versus
KC mice
LSL-Kras
G12D/+
;Pdx-1-Cre (KC) mice 20/42 (48%) N/A
Polyoma-middle T (PyMT) mice 16/20 (80%) 0.027
C57BL/6 WT mice 12/12 (100%) 0.0007
Various strains of mice were injected i.p. with LPS (4 mg/kg) once a week for
4 weeks. The percentage of survivors one week after the nal LPS injection is
shown. WT, wildype.
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1172 | Carcinogenesis, 2016, Vol. 37, No. 12
cytokine assay or by enzyme-linked immunosorbent assays (ELISAs). In pro-
tocol 3 (
Figures 3–5 and Supplementary Table3, available at Carcinogenesis
Online), 9-week-old KC mice were fed CDDO-Im or control chow. Two days
later, mice were injected i.p. with LPS once a week for 4 weeks. Four or eight
weeks after the initial LPS challenge, pancreas, spleen and plasma were har-
vested and analyzed by ow cytometry, IHC, a multiplex cytokine assay and
ELISAs. Aseparate cohort of 9-week-old KC mice (n= 7–8 mice per group)
was fed control diet or CDDO-Im in diet for 4 weeks, and immune cells in the
pancreas and spleen were detected by IHC (
Figure3E andF).
Flow cytometry
Half of the pancreas and spleen removed from KC mice were minced and
incubated separately in digestion media consisting of collagenase (300 U/ml,
Sigma), dispase (1 U/ml, Worthington) and DNAse (2 U/ml, Calbiochem)
for 30 min at 37°C with stirring. Cells were then passed through a 40-μm
Cell Strainer (BD Falcon), and RBC eliminated with lysing solution (eBio-
science). Single cells were resuspended in a solution of phosphate-buff-
ered saline/0.5% bovine serum albumin/0.1% azide and stained 1 h at 4°C
with the following antibodies: CD45-VioGreen, Gr-1-PE, CD11b-FITC (all
Miltenyi, 3μg/ml) and 5μg/ml antimouse CD16/CD32 antibody (clone 93,
eBioscience) to reduce antibody binding to Fc receptors. Propidium iodide
staining was used to exclude dead cells. Cells were analyzed using an
eight-color MACSQuant VYB (MiltenyiBiotec) with three laser sources (405,
488 and 561 nm) and FlowJo x.10.0.7r2 software (Tree Star).
IHC
The other half of the pancreas and spleen removed from KC mice were
xed in 10% phosphate-buffered formalin for at least 48 h, embedded in
parafn blocks and sectioned (5–6 µm). Hydrogen peroxide was used to
quench endogenous peroxidase activity. Sections were immunostained
with CD45 (1:100 eBiosciences) and biotinylated antirat secondary (Vector)
or biotinylated Gr1 (1:50 R&D) antibodies. Signal was detected using a
Vectastain ABC kit and DAB substrate (Vector) following the manufactur-
er’s recommendations. Sections were counterstained with hematoxylin
(Vector). To quantify the % of immune cells in the pancreas, the number
of reactive ducts and PanINs containing CD45+ cells was counted in sec-
tions from six mice per group in a blinded fashion by two operators. In the
spleen, slides were randomized and coded before the % of Gr1+ cells was
calculated on four sections per slide from four mice per group.
Multiplex cytokine assay andELISAs
Plasma from KC mice was aliquoted and stored at −80°C until use. Cytokine
levels in plasma were measured using a Millipore mouse 32plex kit (EMD
Millipore). Calibration curves from recombinant cytokine standards were
prepared with 3-fold dilution steps. Standards and spiked controls were
measured in triplicate, samples were measured once and blank values
were subtracted from all readings. Assays were carried out in a 96-well
ltration plate (Millipore) at room temperature, following the manufactur-
er’s protocol. The uorescence intensity of the Luminex beads was meas-
ured using a Bio-Plex array reader, and Bio-Plex Manager software with
ve-parametric curve tting was used for data analysis. Levels of specic
cytokines in plasma and pancreas extract were also analyzed by ELISAs
(R&D Systems), according to the manufacturer’s protocol.
Statistical analysis
Results are expressed as the mean ± SE and were analyzed by t-test or a
Mann–Whitney Rank Sum test if the data did not t a normal distribution.
For experiments with more than two groups, data was analyzed by one-
way ANOVA followed by a Tukey test, or one-way ANOVA on ranks and
Dunn’s test (SigmaStat 3.5). Categorical data was analyzed by the Fisher
exact test. P<0.05 was considered statistically signicant.
Results
KC mice are more sensitive to LPS than WTmice
Daniluk etal. (
11) recently reported that inammatory stimuli
form a positive feedback loop with oncogenic Ras, prolonging
Ras signaling and thus accelerating carcinogenesis in the pan-
creas. Because infections can cause chronic inammation, a
Figure1. LPS increases the inltration of CD45+ cells into the pancreas and Gr1+
myeloid derived suppressor cells (MDSC) into the spleen of Kras
G12D
;Pdx1-Cre
(KC) mice. (A) Nine-week-old KC mice were injected i.p. with saline (vehicle) or
LPS (4 mg/kg) once a week for 4 weeks. Four or 8 weeks after the initial LPS chal-
lenge, the pancreas and spleen were harvested and analyzed. The % of CD45+
immune cells and the % of CD45+ macrophages (CD11b+GR1−) and MDSCs
(CD11b+GR1+) in the pancreas and spleen were analyzed by ow cytometry (B,
C) and by immunohistochemistry (IHC in D, E). Representative histograms are
shown in B.In (C), **P=0.057 versus saline for CD45+ cells in the pancreas and
*P=0.036 versus saline for GR1+ cells in the spleen; n=9 paired mice per group.
Magnication=200× in (D) and (E).
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A.S.Leal et al. | 1173
major risk factor for pancreatitis and pancreatic cancer, they
injected mice with 10 mg/kg LPS, once a week for 4 weeks. This
experimental paradigm increased early PanIN lesions in mice
with an activating Kras mutation in the acinar cells of the pan-
creas but had no effect in WT mice (11). In KC mice, Kras muta-
tions are targeted to the pancreas by the Pdx1-Cre promoter. The
Pdx1 transcription factor is expressed in all pancreatic precursor
cells during development, but its expression is limited to islet
cells in the adult mouse (6). When we used the same protocol of
LPS injections in KC mice, this dose was unexpectedly toxic, as
83% (10/12) of the KC mice died within 2 weeks of the initial LPS
injection and the majority of these mice died within 48 h of the
LPS challenge (data not shown). When the LPS dose was reduced
to 4 mg/kg (Table1), only 48% (20/42) KC mice survived the LPS
challenge. Notably, 100% (12/12) of WT mice on the same genetic
background (C57BL/6) as the KC mice survived the lower dose of
LPS (P<0.05 versus KC mice). In PyMT mice, also on a C57BL/6
background, expression of the polyomavirus middle T-antigen
(PyMT) gene is regulated by the MMTV promoter. These mice
are widely used to study the role of the immune system in
cancer and demonstrate the importance of tumor associated
macrophages in the progression of breast cancer. Interestingly,
80% (16/20) PyMT mice lived after the LPS injections, suggesting
the inammation that accompanies tumor development in this
strain makes them more susceptible to LPS than WT mice but
not as susceptible as the KC mice.
LPS increases the percentage of CD45+ immune cells
in the pancreas and Gr1+ MDSCs in the spleen of
KCmice
Although tumor immunity may be undermined from initiation
in pancreatic cancer (
9), we hypothesized that the inammatory
response to LPS would accelerate the inltration of immune
cells. To characterize the immune response, 9-week-old KC mice
were injected with 4 mg/kg LPS, once a week for four consecutive
weeks (
Figure1A). Four or eight weeks after the nal injection of
LPS, the pancreas and spleen were harvested. Half of each tis-
sue was used for ow cytometry (
Figure1B) and the other half
for IHC. CD45, also known as the leukocyte common antigen,
is expressed on all leukocytes. Despite inherent variability in
vivo, the percentage of CD45+ cells detected by ow cytometry
was markedly higher (P=0.057) in the pancreas of mice injected
with LPS compared to mice injected with saline for nine pairs
of littermate-matched mice (Figure 1C). If additional matched
pairs of KC mice were included (16–20 weeks after the initial LPS
injections), 52.4 ± 3.2% of the cells in the pancreas were CD45+ in
the saline group versus 62.3 ± 4% (P=0.027) in mice challenged
with LPS (data not shown). The percentage of CD45 + CD11b +
Gr1+ MDSC was also signicantly (P<0.05) higher in the spleen
of LPS-treated mice; MDSCs are immunosuppressive and block
the antitumor activity of T cells in PDAC (26,27). In the mice
injected with LPS, 17.6 ± 2% of the CD45+ cells in the spleen were
Gr1+ compared to only 9.8 ± 2.2% of the cells in mice injected
with saline. No changes were detected in the % of CD45+ cells
in the spleen, Gr1+ MDSC in the pancreas, or CD45 + CD11b +
Gr1− macrophages in the pancreas or spleen (data not shown).
To conrm these results, tissue were also analyzed by IHC. In
the pancreas, CD45+ staining was higher in sections from the
LPS-treated mice than in the saline group (Figure1D). Notably,
CD45+ immune cells were concentrated around early PanIN
lesions, which increased in number and severity over time. In
the spleen (Figure1E), Gr1+ MDSC were found in red pulp, not in
the T lymphocytes found in white pulp and Gr1+ staining was
higher in the groups treated withLPS.
Notably, the immune cell population in the pancreas and
spleen of KC mice is different than in WT mice, even without
LPS stimulation. As shown in Supplementary Figure 1, avail-
able at Carcinogenesis Online, there is a signicantly (P < 0.05)
higher percentage of both CD45+ cells in the pancreas and
Gr1+ MDSC in the spleen of KC mice than in WT mice. However,
LPS can enhance the inltration of CD45+ and Gr1+ cells in KC
mice (
Figure 1C–E). To conrm that LPS also accelerated the
Figure2. CDDO-Im decreases acute inammatory cytokine production in mice challenged with LPS. (A) Nine-week-old KC (LSL-Kras
G12D/+
;Pdx-1-Cre) or WT (wildtype
LSL-Kras
G12D/+
or Pdx-1-Cre) mice were fed CDDO-Im (100 mg/kg diet) or control diet 2days prior to being challenged with LPS. Mice were then injected i.p. with a single
dose of saline (vehicle) or LPS (4 mg/kg). Twenty-four hours later, plasma (B, C) and pancreas (D) were harvested and analyzed with a multiplex cytokine assay (Sup-
plementary data, available at Carcinogenesis Online) or by ELISAs (B–D). n=8–12 mice per group. In (B) *P=0.021 WT versus KC mice and *P<0.05 KC LPS versus KC LPS
+ Im in (C) and (D).
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1174 | Carcinogenesis, 2016, Vol. 37, No. 12
development of PDAC in KC mice, the number of pancreatic
lesions was evaluated on H & E stained slides. Changes in CD45+
and MDSC populations were observed 4 or 8 weeks after the
LPS challenge (Figure1), but there was not sufcient pathology
at these time points to detect differences between groups. At
12 weeks, however, there was a trend toward higher numbers
of PanINs in the LPS group (49.4 ± 8.1 per pancreas) than in the
saline group (41.8 ± 13.1 per pancreas). The number of PDACs
per section was signicantly (P=0.03) higher in the pancreas of
mice treated with LPS (3.14 ± 0.41) than with saline (1.98 ± 0.15;
n=5 mice per group).
Cytokine proles differ in KC mice versus WT mice
challenged withLPS
Because of the importance of inammation in the pathogenesis
of pancreatic cancer (
28,29), we compared the cytokine proles
of KC and WT mice 24 h after a single injection of either LPS
or saline (Figure2A). Plasma from 6 to 7 mice per group was
analyzed with a multiplex assay (
Supplementary Table1, availa-
ble at Carcinogenesis Online), which is useful for measuring mul-
tiple cytokines in a single sample. The only difference between
WT and KC mice injected with saline was a markedly higher
level of G-CSF, or CSF3, in the KC mice (2116 ± 498 pg/ml) versus
WT mice (828 ± 76 pg/ml; P=0.052), suggesting that an activat-
ing mutation in Kras mutation is sufcient to increase G-CSF
production. Because G-CSF mediates the expansion and recruit-
ment of MDSCs and enhances angiogenesis and metastases (30),
high expression of G-CSF is an indicator of poor prognosis in
cancer patients. An ELISA was used to conrm these results, and
as shown in Figure2B, endogenous G-CSF levels were more than
4-fold higher in the KC mice than in WT mice (P=0.021; n=8
per group).
LPS increased the secretion of 21 cytokines in WT mice and
29 cytokines in KC mice. Notably, cytokine levels were clearly
higher in the KC mice than in the WT mice challenged with LPS
for almost all of these cytokines. Because of variability between
Figure3. CDDO-Im protects against toxicity induced by LPS and decreases the inltration of CD45+ immune cells into the pancreas and Gr1+ MDSC into the spleen
of KC mice. (A) Nine-week-old KC mice were fed CDDO-Im (100 mg/kg diet) 2days prior to an LPS challenge. Mice were then injected i.p. with saline (vehicle) or LPS
(4 mg/kg) once a week for 4 weeks. (B) The percentage of survivors 1 week after the nal LPS injection. Four (C) or eight (D) weeks after the initial LPS challenge, the
pancreas and spleen were harvested and analyzed by ow cytometry. In (E, F), KC mice were fed CDDO-Im or control diet for 4 weeks but not challenged with LPS.
Pancreas and spleen were harvested 4 weeks later to evaluate immune cell populations. n=8–10 pairs per group; *P<0.05 versus control; ***P<0.001 versus control.
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