SUPPLEMENTARY INFORMATION
Title: Myeloid-derived suppressor cells play a central role in attenuated Listeria
monocytogenes-based immunotherapy against metastatic breast cancer in young and
old mice
Authors: Dinesh Chandra$1, Arthee Jahangir$1, Wilber Quispe-Tintaya1, Mark H.
Einstein2, and Claudia Gravekamp*1
1
Albert Einstein College of Medicine Department of Microbiology and Immunology,
1300 Morris Park Avenue, Room F407A, Bronx, NY 10461;
2
Albert Einstein College of Medicine Department of Obstetrics & Gynecology and
Women’s Health; Division of Gynecologic Oncology 1695 Eastchester Road, Room
601 Bronx, NY 1046
$Both
authors equally contributed to the manuscript
* To whom correspondence should be addressed. Email: claudia.gravekamp@einstein.yu.edu
To whom correspondence should be addressed
* Claudia Gravekamp, PhD
Albert Einstein College of Medicine
Department of Microbiology and Immunology
1300 Morris Park Avenue
Forchheimer Bldg, Room 407A
Bronx, NY 10461
Email: claudia.gravekamp@einstein.yu.edu
Phone: 718-430-4048(office)/4067 (lab)/Fax: 718-430-8711
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The PDF file includes:
Figure S1: Listeriaat multiplied in metastases and primary tumor but not in normal
tissues in vivo
Figure S2: Listeriaat did not multiply in MDSC of non-tumor-bearing mice
Figure S3: Listeriaat infection induced IL-12 production in human MDSC in vitro
Figure S4: Listeriaat was highly effective against metastases in pancreatic cancer model
Panc-02
Figure S5. Therapeutic treatment with Listeriaat was equally effective against breast
cancer metastases in young and old mice
Figure S6. Gating of MDSC in blood and primary tumors of young and old mice
Figure S7: Listeriaat reduced the absolute numbers of MDSC in blood of the 4T1 model
TEXT
Infection and kill of normal cells by Listeriaat in vitro
MATERIALS AND METHODS includes:
Therapeutic immunization protocol 4T1 model
Semi-therapeutic immunization protocol Panc-02 model
Isolation of Listeriaat from tumors, metastases, and normal tissues
Listeriaat infection of MDSC from mice without tumors
Detection of IL-12 in human MDSC by flow cytometry
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TEXT
Listeriaat infects and kills normal cells in vitro. In a previous study we have shown that
Listeriaat infects and kills primary cultures of BJ (human fibroblasts) and MEF (mouse
fibroblasts) with high efficiency (Kim et al, 2009), which is here described in more
detail. 5 x 105 cells/ml were infected with 108 (per well) of Listeriaat, for 1 hr at 37°C in
culture medium. After the incubation with gentamicin (50 g/ml) for 1 hr (killing all
extracellular Listeria bacteria), cells were washed with PBS, lysed in sterile water, and
serial dilutions were plated onto LB agar to determine the infection rate the next day.
Death of the fibroblasts induced by Listeriaat was determined in vitro as follows. 3x103 of
the fibroblast cells plated in 96 well plates were infected with 106 (per well) of Listeriaat,
for 3 hrs at 37°C. Gentamicin (50 g/ml) was added until live and dead cells were
counted using Trypan blue staining on the next day.
LEGENDS
Figure S1: Listeriaat multiplied in metastases and primary tumor but not in normal
tissues. Here, we analyzed the behavior of Listeriaat in BALB/cByJ mice with or without
4T1 tumors. Mice with or without 4T1 tumors were injected once with 0.5x107 Listeriaat
and analyzed for the number of live Listeriaat in primary tumor, metastases, and in
various normal tissues at various time intervals. In mice with tumors, Listeriaat multiplied
in metastases and primary tumor but not normal tissues, with an exception for the liver
and spleen, because metastases already spread to these organs. In mice without tumors,
no multiplication of Listeriaat was observed in the normal tissues. In both, mice with or
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without tumors, Listeriaat was cleared in all normal tissues within 7 days, while they
survived in the metastases and less pronounced in the primary tumor. n=3 mice per
group. Mice were individually analyzed and the results were averaged. The graphs are a
representative of three experiments. The error bars represent the SEM.
Figure S2: Listeriaat did not multiply in MDSC of non-tumor-bearing mice. Splenic
MDSC of non-tumor bearing BALB/cByJ mice were isolated and cultured with Listeriaat
at a 1:10 ratio for 1 hr, then gentamicin was added, and terminated at different time
points. The infected MDSC were lysed in water and plated onto LB agar to determine the
number of Listeriaat CFU the next day. n=3 mice per group. Results of two experiments
were averaged. The error bars represent the SEM.
Figure S3: Listeriaat infection induced IL-12 production in human MDSC in vitro.
White blood cells were isolated from patients with cervical intraepithelial neoplasia
(CIN2/3) or with cervical cancer, and then infected with Listeriaat (MDSC:Listeriaat
=1:10 ratio) for 1hr, cultured for 24 hrs in the presence of gentamicin, and then analyzed
for the intracellular production of IL-12 by flow cytometry. This experiment was
performed with blood of 8 patients, and the results were averaged. Unpaired t-test p<0.05
is statistically significant. The error bars represent the SEM. The human MDSC are
CD11b+CD33+CD14+CD15+HLA-DR-.
Figure S4: Listeriaat was highly effective against metastases in a pancreatic cancer
model Panc-02. Semi-therapeutic immunizations with Listeriaat dramatically reduced the
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number of metastases in the Panc-02 model. C57Bl/6 mice were immunized with
Listeriaat i.p. (0.5x107 cfu) one time before and two times after tumor development on
days 0, 7, and 14, and challenged with Panc-02 tumor cells (2x106 cells) in the mammary
fat pad on day 3. All mice were euthanized on day 21, and analyzed for the location and
number of metastases (A) and for tumor weight (B). n=5 mice per group. This experiment
was performed three times and the results were averaged. Unpaired t test p<0.05 is
statistically significant. The error bars represent the SEM.
Figure S5: Therapeutic treatment with Listeriaat was equally effective against breast
cancer metastases in young and old mice. An optimal therapeutic immunization
protocol with Listeriaat was developed in young mice with metastatic breast cancer.
Young BALB/cByJ mice were injected with 104 4T1 tumor cells on day 1, and
immunized with various doses of Listeriaat at different time intervals all starting on day 3
(three immunizations with 0.5x107 Listeriaat CFU once a week, or five immunizations
with 104 Listeriaat CFU every other day) (A). n=5 mice per group. All mice were
euthanized on day 16 and analyzed for the number of metastases and tumor weight.
Representative of two experiments. Unpaired t test. *p<0.05, **<0.01, ***<0.001,
****<0.0001 is significant. The error bars represent the SEM. The optimal therapeutic
immunization protocol was then tested in young and old BALB/cByJ mice with
metastatic breast cancer (4T1 model) (B). All mice were euthanized on day day 16 and
analyzed for the number of metastases and tumor weight. n=5 mice per group. Average of
three experiments. Unpaired t test. *p<0.05, **<0.01, ***<0.001, ****<0.0001 is
significant. The error bars represent the SEM.
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Figure S6. Gating of MDSC in blood and primary tumors of young and old mice.
Semi-therapeutic immunizations with Listeriaat significantly reduced the percentage of
gMDSC in blood of both young and old mice. Mice were immunized with Listeriaat on
days 0, 7, and 14, and challenged with 4T1 tumor cells on day 4. All mice were
euthanized on day 16 and analyzed for MDSC. In blood, the total MDSC population
(CD11b+Gr1+)(top and bottom box), the gMDSC population (CD11b+Gr1high)(top box),
and mMDSC population (CD11b+Gr1low)(bottom box), were gated within the total live
leukocyte population and analyzed by flow cytometry. In primary tumors, the total
MDSC
population
(CD11b+Gr1+)(top
and
bottom
box),
gMDSC
population
(CD11b+Gr1high)(top box), and mMDSC population (CD11b+Gr1low)(bottom box), were
gated within the total live CD45+ population of the tumor cell suspension, and analyzed
by flow cytometry. The granulocytic and monocytic character of the CD11b +Grhigh and
CD11b+Gr1low cells, respectively, was confirmed by sorting both fractions using FACS
and stained the sorted cells with Giemsa.
Figure S7: Listeriaat reduced the absolute numbers of MDSC in blood of the 4T1
model. All mice were immunized with Listeriaat, challenged with 4T1 tumor cells, and
euthanized on day 16. The total MDSC population (CD11b+Gr1+), as well as the gMDSC
population (CD11b+Gr1high) and mMDSC population (CD11b+Gr1low), were gated within
the total live white blood cell population, and analyzed by flow cytometry (see Fig S7).
The absolute numbers of MDSC were calculated per 1 ml of blood. Briefly, the % of
MDSC was divided by 100 and then multiplied with the total number of cells per ml of
blood for each sample. Graphs are the average of 3 experiments (mice were individually
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analyzed). n=5 mice per group. Unpaired t test. *p<0.05, **<0.01, ***<0.001,
****<0.0001 is significant. The error bars represent the SEM.
MATERIALS AND METHODS
Therapeutic immunization protocol 4T1 model. Mice were immunized and challenged
with tumor cells as described previously with some modifications (Kim et al, 2009).
Briefly, a BALB/cByJ background was used for the 4T1 model. Five therapeutic
immunizations with 104 CFU of Listeriaat (LD50=108 CFU) per 500 μl saline, or saline
alone were administered intraperitoneally (i.p.), starting on day 3 after tumor challenge
(0.5 x 105 4T1), and administering the immunizations every other day thereafter. The
mice were euthanized 2 days after the last immunization, and analyzed for tumor weight,
frequency and location of metastases. All untreated 4T1 mice developed a primary tumor
that extended to the chest cavity lining and metastasized predominantly to the mesenteric
lymph nodes (MLN), and less frequently to the diaphragm, portal liver, spleen, and
kidneys within 14 days, as described previously (Kim et al, 2008). Metastases were
visible as nodules and therefore, counted by eye.
Semi-therapeutic immunization protocol Panc-02 model Mice were immunized and
challenged with tumor cells as described previously with some modifications (Kim et al,
2009). Briefly, a C57Bl/6 background was used for the Panc-02 model. Immunizations
with 0.5x107 cfu of Listeriaat (LD50=108 CFU) per 500 μl saline, or saline alone were
administered intraperitoneally (i.p.) on days 0, 7, and 14, and Panc-02 tumor cells (2x106
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cells) were injected into the mammary fat pad on day 3. All mice were euthanized on day
21 and analyzed for the number of metastases and tumor weight. In the untreated mice, a
primary tumor developed in the mammary fat pad and extended to the chest cavity lining,
but primary tumors stayed relatively small, and metastasized (also visible by the naked
eye as nodules and counted) predominantly to the portal liver, resulting in the production
of ascites in the peritoneal cavity, and to the mesenteric lymph nodes (MLN), and less
frequently to the diaphragm, spleen, and kidneys within 10-20 days.
Isolation of Listeriaat from tumors, metastases, and normal tissues. Mice with 4T1
metastases and tumors were immunized once with Listeriaat (0.5x107 CFU), and
euthanized at various time points. Metastases from all locations (pooled), tumors and
normal tissues (spleen, liver, lung, heart, kidneys, and blood) were dissected and treated
with gentamicin for 1 hr to kill all extracellular bacteria, and then washed to remove the
dead bacteria and gentamicin. Subsequently, metastases, tumors and spleens were
homogenized, plated on agar, and counted for Listeriaat colonies the next day. The
number of Listeriaat was calculated per gram tissue.
Listeriaat infection of MDSC from mice without tumors. mMDSC and gMDSC were
isolated from spleens of mice without 4T1 tumors, and infected with Listeriaat in a 1:10
ratio, then cultured for 1hr in RPMI containing 10% FBS. Subsequently the infected cells
were treated with gentamicin (50 g/ml) for 1 hr to kill all extracellular bacteria, and
washed to remove the dead bacteria and gentamicin. Subsequently, the infected cells
were cultured and stopped at various time points. To measure the number of live Listeriaat
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bacteria, the infected cells were lysed in water, plated on agar and counted for Listeria at
colonies the next day. The number of Listeriaat CFU was calculated per 106 cells.
Detection of IL-12 in human MDSC by flow cytometry. Following the institutional
review board approval from Montefiore Medical Center, informed consent was obtained
from all participants. Blood was obtained from women with cervical intraepithelial
neoplasia (CIN2/3) or with cervical cancer, between 31 and 66 years old. White blood
cells were obtained by mixing one volume of blood with one volume of Dextran 3%, and
kept at room temperature for 60 min. After centrifugation the white blood cells were
obtained from the top layer, and incubated with lysis buffer to eliminate remaining red
blood cells as described previously (Castro et al, 2009). Subsequently, the white blood
cell population was and infected with Listeriaat in a 1:10 ratio and cultured for 1hr in
RPMI containing 10% FBS. Then, the infected MDSC were treated with gentamicin (50
g/ml) until the end of the experiment (24 hrs) to kill all extracellular bacteria. After the
24hrs, the cells were incubated with Golgi-plug for 6 hrs, and stained with anti-CD11b, CD33, -CD14, -CD15, and -HLA-DR antibodies to identify the human MDSC, and
subsequently stained for the intracellular production of IL-12 with anti-IL-12 antibodies.
The samples were analyzed with a Becton Dickinson LSRII digital bench top flow
cytometer [12-color, 4-laser system]. All antibodies were purchased from BD
Biosciences.
Statistical analysis. To statistically analyze the effects of Listeriaat on the growth of
metastases and tumors and immune responses in the 4T1 model or Panc-02 model, the
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unpaired t test and the analysis of variance (ANOVA) (one way) were used. Values
p<0.05 were considered statistically significant. *p<0.05, **<0.01, ***<0.001,
****<0.0001 is significant.
REFERENCES
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Kim S, Castro F, Gonzalez D, Maciag P, Paterson Y, Gravekamp C (2008) Mage-b
vaccine delivered by recombinant Listeria monocytogenes is highly effective against
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Kim S, Castro F, Paterson Y, Gravekamp C (2009) High efficacy of a Listeria-based
vaccine against metastatic breast cancer reveals a dual mode of action. Cancer Res
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