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Supplementary Materials for
Two-step enhanced cancer immunotherapy with engineered
Salmonella
typhimurium secreting heterologous flagellin
Jin Hai Zheng, Vu H. Nguyen, Sheng-Nan Jiang, Seung-Hwan Park,
Wenzhi Tan,
Seol Hee Hong, Myung Geun Shin, Ik-Joo Chung, Yeongjin Hong,
Hee-Seung Bom,
Hyon E. Choy, Shee Eun Lee, Joon Haeng Rhee,* Jung-Joon Min*
*Corresponding author. Email: [email protected] (J.-J.M.);
[email protected] (J.H.R.)
Published 8 February 2017, Sci. Transl. Med. 9, eaak9537
(2017)
DOI: 10.1126/scitranslmed.aak9537
This PDF file includes:
Materials and Methods
Fig. S1. NF-κB activation by LPS and FlaB in cancer cells and
peritoneal
macrophages in vitro.
Fig. S2. Luciferase assay in HCT116 cancer cells.
Fig. S3. Spleen weight after Salmonella treatment.
Fig. S4. Analysis of cell populations in the spleen after
Salmonella treatment.
Fig. S5. Noninvasive monitoring of bacterial distribution in
vivo.
Fig. S6. Distribution of bacteria in MC38 tumor–bearing
mice.
Fig. S7. Detection of bacteria and FlaB in liver and tumor
tissues.
Fig. S8. Systemic toxicity of FlaB-expressing bacteria.
Fig. S9. Photographs of mice treated with FlaB-secreting
bacteria.
Fig. S10. Antitumor effect in a B16F10 melanoma model.
Fig. S11. Tumor growth in WT and knockout mice.
Fig. S12. Effect of bacterial treatments on tumor growth in TLR4
knockout mice.
Fig. S13. Cell infiltration in WT and knockout mice after
Salmonella treatment.
Fig. S14. Macrophage polarization after treatment with
FlaB-secreting bacteria
assessed by quadruple staining.
Fig. S15. Detection of tumor-suppressive cytokines in tumor
tissues.
Table S1. Bacterial strains and plasmids used in the study.
Table S2. Antibodies used in the study.
www.sciencetranslationalmedicine.org/cgi/content/full/9/376/eaak9537/DC1
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Materials and Methods
Cell lines
MC38 murine colon carcinoma cells were kindly provided by Dr.
Je-Jung Lee (Chonnam National
University, Republic of Korea). B16F10 mouse melanoma cell line
was obtained from the American
Type Culture Collection (ATCC, CRL-6475). A human colon
carcinoma cell line stably expressing
firefly luciferase (HCT116-luc2) was purchased from Perkin Elmer
(Product No. 124318). MC38
and B16F10 were maintained in high-glucose Dulbecco’s Modified
Eagle’s Medium, and HCT116-
luc2 in McCoy's 5a Modified Medium, supplemented with 10% fetal
bovine serum and 1%
penicillin-streptomycin. The cells were authenticated by the
Waterborne Virus Bank (Seoul,
Republic of Korea).
Sample preparation and Western blot analysis
To check FlaB expression in vitro, S. typhimurium (SL) ΔppGpp
was transformed with the pFlaB
plasmid. Fresh bacterial cultures (1 h) were grown to A600 = 0.5
to 0.7 before addition of 0% or 0.2%
L-arabinose. After 3 h, samples were collected and centrifuged
to separate the bacterial pellet. The
remaining culture medium was then filtered (Supernatant).
Bacterial protein (20 μg) or purified V.
vulnificus FlaB (0.1 μg) was used for immunoblot analysis. The
expression and secretion of FlaB
protein (43 kDa) were confirmed with an anti-FlaB antibody
(1:20,000, absorbed polyclonal
antibody) (25). To check NF-κB activation in cancer cells and
peritoneal macrophages, cells were
overnight cultured in 1% FBS medium for starvation to decrease
background signal. After treatment
with 100 ng/ml LPS or 100 ng/ml FlaB at the indicated time
points, samples were collected, and
nuclear fractions were obtained using a nuclear extract kit
(Active Motif).
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Protein concentration was measured in a BCA assay (Thermo),
protein samples were separated in 12%
sodium dodecyl sulfate-polyacrylamide gels. The protein was then
transferred to nitrocellulose
membranes (Bio-Rad) and blocked with 5% skim milk for 2 h at
room temperature. The membranes
were then probed with a specific primary antibody (table S2),
followed by a horseradish peroxidase-
conjugated secondary antibody (table S2). Immunoreactive
proteins were detected using Luminol
reagents (Santa Cruz Biotechnology) and visualized using a Fuji
Film image reader (LAS-3000; Fuji
Film). Purified recombinant V. vulnificus FlaB was used as a
positive control.
Luciferase reporter assay
HCT116 cells were transfected with NF- κB reporter plasmids
(pNF-κB-Luc) with or without 3X
Flag-TLR5-expressing plasmid (p3XFlag-hTLR5) using Effectene
Transfection Reagent (Qiagen) as
previously reported (25). Luciferase activity was normalized to
LacZ expression using the control
expression plasmid pCMV-β-Gal (BD Biosciences). At 24 hours
after the transfection, cells were
incubated with PBS or FlaB protein (100 ng/well) for 6 hours.
Cells were lysed with a lysis buffer
(Promega), and the luciferase activity was measured by a
luminometer (MicroLumatPlus LB 96V;
Berthold). Luciferase activity is expressed as fold difference
in activation of FlaB-stimulated samples
relative to PBS-treated samples.
Treatment schedule
Treatments were given on Day 8 after tumor implantation (tumor
volume, ~120 mm3). Tumor-
bearing mice were divided into six treatment groups: (i) PBS
alone, (ii) purified FlaB, (iii)
Salmonellae carrying an empty vector, (iv) FlaB-expressing
Salmonellae without L-arabinose
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induction, (v) Salmonellae plus purified FlaB, and (vi)
FlaB-secreting Salmonellae plus L-arabinose
induction. Bacteria were prepared at a dose of 1 × 107 CFU in
100 μl PBS, as previously described
(5). Purified recombinant V. vulnificus FlaB protein (25) was
administered at a dose of 2 μg in 10 μl
PBS. Each mouse received a single dose of bacteria or PBS via
the lateral tail vein. Purified FlaB
protein was administered every 2 days by intratumoral injection
using a micro injector (Hamilton)
capped with a PrecisionGlide needle (BD). Expression of
therapeutic genes was induced by daily
intraperitoneal administration of 0.12 g L-arabinose from 3 dpi.
Mice receiving combination therapy
with Salmonellae carrying an empty vector plus purified FlaB
received FlaB from 3 dpi.
Viable bacterial counts
To quantify the number of bacteria within the tumor tissue,
tumors were excised from mice and
homogenized in PBS. Samples were serially diluted (10-fold) and
plated on ampicillin-containing
LB plates. After overnight incubation at 37°C, the bacterial
titer (CFU/g tissue) was determined by
counting colonies and calculated with dilution factors and
tissue weight (29).
Quantitative RT-PCR
At 6 h after L-arabinose induction (3 dpi), tumor tissues were
collected and treated with RNAprotect
Bacteria Reagent (Qiagen). Bacterial mRNA was then obtained from
the pellet using the RNeasy
Mini Kit (Qiagen), according to the recommended protocol. cDNA
was generated from 1 μg total
mRNA using an oligo (dT) primer (Promega) and Improm-II Reverse
Transcriptase (Promega).
Quantitative RT-PCR was performed using CYBR green (Takara) and
the following RT-PCR primer
sets: S. typhimurium housekeeping gene (aroC): F, TCG CCG ATC
TCC ACG CCT TT, and R,
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GCG CGA AAG TGA CGG TGA TG; and flaB primers: F, GAG CGT CTG TCT
TCA GGT T, and
R, GTT GTA GGA TGT TGG TGG TC. PCR was performed in a Rotor-Gene
machine (Corbett
Research) under the following conditions: 10 min at 95°C,
followed by 40 cycles at 95°C for 15 s,
60°C for 30 s, and 72°C for 15 s. The amount of flaB mRNA was
determined by comparison with
that of the housekeeping gene Salmonella aroC (data expressed as
the fold difference).
NO colorimetric assay
The amount of NO in the tumors was assessed indirectly by
measuring nitrites and nitrates using the
Nitric Oxide Colorimetric Assay Kit (Abcam), according to the
manufacturer’s instructions. At 24 h
after L-arabinose induction, tumor samples were collected and
homogenized in ice-cold lysis buffer
(1:4 w/v) containing a proteinase inhibitor. After 1 h of
incubation on ice, the clarified supernatant
was collected by centrifugation (10,000 g 4°C for 10 min), and
the total protein concentration was
determined by a BCA assay. The clarified samples were
deproteinated using a 10 kDa cutoff filter
(Abcam) to improve NO stability and then kept at ˗80°C. Samples
and standards were exposed to
nitrate reductase and its enzyme cofactor for 1 h at room
temperature to transform nitrate to nitrite.
The enhancer and Griess reaction reagents were then used to
convert nitrite to a purple azo
chromophore compound, and the color was allowed to develop for
10 min. This provided a lower
limit of detection of 1 µM at 540 nm according to a linear
model. The amount of NO was normalized
to the amount of protein (mg).
Immunofluorescence analysis
For immunofluorescence analysis, tumor tissues were collected
from mice and fixed in 4%
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paraformaldehyde for 2 h at 4°C. The tissues were then washed in
PBS, transferred to a 30% sucrose
solution, and incubated overnight at 4°C. Fixed tissues were
embedded in OCT compound and kept
at ˗80°C. Samples were then sectioned (6 µm thickness) using a
microtome (Thermo Scientific) and
mounted on glass slides. The slides were blocked with 5% BSA and
then incubated with primary
antibodies (see table S2) overnight at 4°C. The sections were
then washed and incubated with
secondary antibodies (see table S2) for 1 h at room temperature.
After counterstaining with DAPI
(1:10,000, Invitrogen), the sections were mounted in
ProlongAntifade mounting solution (Invitrogen)
and examined under a FV1000D confocal laser scanning microscope
(Olympus). Images were
analyzed using the FV10-ASW2.0 Viewer software (Olympus).
Optical bioluminescence imaging
Bioluminescence imaging of tumors was performed using an IVIS
100 (Caliper) after intraperitoneal
injection of 750 μg D-luciferin, as described previously
(18).
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Supplementary figures:
Fig. S1. NF-κB activation by LPS and FlaB in cancer cells and
peritoneal macrophages in vitro.
Samples were immunoblotted with antibodies against activated
NF-κB (p-p65), total NF-κB (p65)
(Cell Signaling Technology), and β-actin (Santa Cruz
Biotechnology).
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Fig. S2. Luciferase assay in HCT116 cancer cells. HCT116 cells
were transfected with NF- κB
reporter plasmids (pNF-κB-Luc) with or without 3X
Flag-TLR5-expressing plasmid (p3XFlag-
hTLR5), and stimulated with PBS or FlaB protein (100 ng/well).
Luciferase activity is expressed as
fold difference in activation of FlaB-stimulated samples
relative to PBS-treated samples (n = 5).
HCT116-TLR5+: HCT116 co-transfected with p3XFlag-hTLR5 and
pNF-κB-Luc. HCT116: HCT116
transfected with pNF-κB-Luc.
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Fig. S3. Spleen weight after Salmonella treatment. BALB/c nude
mice were surgically implanted
with two pieces of HCT116-luc2 tumor stably expressing firefly
luciferase (each measuring 1 mm3).
At Day 4 after surgery, mice were treated with PBS, bacteria
harboring pEmpty (SLpEmpt), or
bacteria harboring pFlaB (SLpFla) with L-arabinose induction
from 3 dpi. All animals were
sacrificed at Day 27 after transplantation, and spleen weight
was measured (n = 7 mice/ group).
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Fig. S4. Analysis of cell populations in the spleen after
Salmonella treatment. Single cells were
isolated from the spleens of MC38 tumor-bearing mice at 4 dpi
with Salmonella carrying an empty
vector or Salmonella secreting FlaB. Total cells in the spleen,
along with the numbers of CD4+
(CD3+CD4+) and CD8+ (CD3+CD8+) cells, DCs (CD11b+CD11c+), and
neutrophils (Gr-1+), are
shown (n = 9 mice/ group).
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Fig. S5. Noninvasive monitoring of bacterial distribution in
vivo. S. typhimurium expressing
bacterial luciferase (ΔppGpp/Lux) was transformed with
FlaB-encoding plasmid (pFlaB) as
described previously (18). (A) Non-invasive monitoring of
bacterial bioluminescence by optical
imaging. (B) Photographs of representative mice. (˗), without
L-arabinose induction; (+), with L-
arabinose induction (n = 9 mice/ group; images are
representative of three individual experiments). L:
liver, T: tumor.
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Fig. S6. Distribution of bacteria in MC38 tumor–bearing mice.
MC38 tumor-bearing mice were
intravenously injected with 1 × 107 CFU engineered
FlaB-expressing Salmonellae. Blood, lung, liver,
spleen, and tumor tissues were collected, and the number of
viable bacteria was counted at the
indicated time points (n = 11 mice/group).
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Fig. S7. Detection of bacteria and FlaB in liver and tumor
tissues. MC38 tumor-bearing mice
were treated with FlaB-expressing bacteria (1 × 107 CFU). At 3
dpi, mice were injected with L-
arabinose (0.12 g) to induce FlaB, then 6 h later, the liver and
tumor tissues were isolated and
prepared for immunofluorescence staining. Sections were stained
with antibodies against Salmonella
(SL) (green) and FlaB (red). Nuclei were stained with DAPI
(blue). A merged image is also shown
(Merged). Data are representative of three independent
experiments. Scale bar = 50 μm.
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Fig. S8. Systemic toxicity of FlaB-expressing bacteria. MC38
tumor-bearing mice (n = 14
mice/group used for three individual experiments) were injected
with PBS, S. typhimurium carrying
an empty vector (SLpEmpty), or S. typhimurium carrying pFlaB
(SLpFlaB), followed by an
intraperitoneal injection of L-arabinose (0.12 g) starting on 0
dpi or 3 dpi. Serum levels of alanine
aminotransferase (ALT), aspartate aminotransferase (AST), blood
urea nitrogen (BUN), creatinine
(CREA), C-reactive protein (CRP), and procalcitonin (PCT) were
measured at 5 dpi. In the box and
whisker plot, the lines at the top and bottom of the boxes
represent the upper and lower quartiles,
respectively. The line within the box represents the median
value. The whiskers mark the 10th–90th
percentiles. Normal values: ALT, 17–77 IU/l; AST, 54–298 IU/l;
BUN, 8–33 mg/dl; CREA, 0.2–0.9
mg/dl; CRP,
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Fig. S9. Photographs of mice treated with FlaB-secreting
bacteria. C57BL/6 mice (n = 20)
bearing MC38 subcutaneous tumors were injected with 1 × 107 CFU
SLpFlaB, followed by
intraperitoneal injection of L-arabinose (0.12 g) from 3 dpi.
Among the 20 mice, the tumor was
completely eradicated in 11 mice (red numbers).
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Fig. S10. Antitumor effect in a B16F10 melanoma model. C57BL/6
mice were subcutaneously
injected with B16F10 melanoma cells (5 × 105). When the tumors
reached a volume of
approximately 120 mm3, mice were treated with PBS, Salmonella
carrying an empty vector
(SLpEmpty), or a FlaB-encoding plasmid (SLpFlaB) followed by
L-arabinose induction (0.12 g)
from 3 dpi. (A) Tumor size. (B) Representative photos of mice
from each group (n = 4 mice/group).
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Fig. S11. Tumor growth in WT and knockout mice. C57BL/6 mice
(WT, TLR4-/-, TLR5-/-, and
MyD88-/-; n = 8 mice/group) subcutaneously bore MC38 tumors.
When the tumors reached a volume
of approximately 120 mm3, mice were treated with PBS. There was
no significant difference in
tumor growth among groups.
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Fig. S12. Effect of bacterial treatments on tumor growth in TLR4
knockout mice. TLR4-/- mice
bearing MC38 tumors were treated with PBS, Salmonella carrying
an empty vector (SLpEmpty), or a
FlaB-encoding plasmid (SLpFlaB) followed by L-arabinose
induction (0.12 g) from 3 dpi. (A)
Tumor growth as a percentage of starting size. There was no
statistical difference between the groups.
(B) Representative photos of mice from each group (n = 8
mice/group). P (PBS vs. SLpEmpty) =
0.3829; P (PBS vs. SLpFlaB) = 0.1375; P (SLpEmpty vs. SLpFlaB) =
0.4452.
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Fig. S13. Cell infiltration in WT and knockout mice after
Salmonella treatment.
Immunofluorescence staining was performed to examine immune cell
infiltration into tumors in WT,
TLR5-/-, and TLR4-/- mice at 3 dpi. MOMA-2: a monoclonal
antibody against monocytes and
macrophages; Neu marker: a monoclonal antibody against
neutrophils. Representative images from
three independent experiments are shown. Scale bar = 50 µm.
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Fig. S14. Macrophage polarization after treatment with
FlaB-secreting bacteria assessed by
quadruple staining. FlaB was induced with L-arabinose at 3 dpi,
and single cells were isolated from
MC38 tumors 24 h later. Samples were quadruple stained for
CD11b, F4/80, CD68, and Ly6C and
analyzed by FACS. All samples were pre-gated on CD11b and F4/80
double-positive cells and then
subsequently gated on the CD68int/Ly6Cint population to identify
the M1-like fraction. The contour
plots clearly show an increased M1-like macrophage population
after treatment with FlaB-expressing
Salmonella (n = 6 mice/ group; data are representative of three
independent experiments).
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Fig. S15. Detection of tumor-suppressive cytokines in tumor
tissues. Tumor tissues (n = 9
mice/group) were isolated from mice 24 h after L-arabinose
induction on Day 4 after infection.
Cytokines were detected by ELISA. (A) IL-1β. (B) TNF-α.
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Table S1. Bacterial strains and plasmids used in the study.
Strains Relevant genotype Plasmids Reference
SHJ2168 ΔrelA, ΔspoT (ΔppGpp) 46
pBAD-PelB-Rluc8 29
pBAD-empty (pEmpty) This study
pBAD-PelB-FlaB (pFlaB) This study
pNF-κB-Luc 25
p3XFlag-hTLR5 25
pCMV-β-Gal BD Biosciences
CFU: colony-forming unit.
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Table S2. Antibodies used in the study.
Antibody name Antibody description Company/Catalog no.
Remark
Rabbit anti-FlaB polyclonal antibody, absorbed Rabbit anti-FlaB
Joon Haeng Rhee
Chonnam National Univ. (25) Primary Ab
Goat anti-Salmonella polyclonal antibody Anti-Salmonella GenWay
Biotech/
GWB-AB2632
Rat anti-mouse F4/80 (A3-1) Rat anti-mouse F4/80
AbDSerotec/MCA497GA
Anti-CD86 antibody Rabbit anti-CD86 Abcam/ab112490
Phospho-NF-KB p65 (Ser536) antibody Rabbit anti-NF-κB p65
(Ser536)
Cell Signaling
Technology/3031
NF-κB p65 (D14E12) XP® rabbit mAb Rabbit anti-NF-κB p65 Cell
Signaling
Technology/8242
β-actin (C4) Mouse anti-β-actin Santa Cruz Biotechnology
/SC-47778
CD206 (C-20) Goat anti-CD206 Santa Cruz Biotechnology
/SC-34577
Monocyte/macrophage marker
(MOMA-2)
Rat mAb anti-monocyte +
macrophage
Santa Cruz Biotechnology
/SC-59332
Neutrophil marker (6A608) Rat mAb anti-neutrophil Santa Cruz
Biotechnology
/SC-71674
Peroxidase-conjugated rabbit anti-mouse
immunoglobulins
Rabbit anti-mouse Dako/P0260 Secondary Ab
Peroxidase-conjugated goat anti-rabbit
immunoglobulins
Goat anti-rabbit Dako/P0448
Alexa Fluor® 488 donkey anti-rat IgG (H+L)
antibody
Donkey anti-rat Invitrogen/A21208
Alexa Fluor® 555 donkey anti-rabbit IgG (H+L)
antibody
Donkey anti-rabbit Invitrogen/A31572
Alexa Fluor® 647 donkey anti-goat IgG (H+L)
antibody
Donkey anti-goat Invitrogen/A21447
Anti-TLR5 Alexa Fluor® 488 mAb (clone19D759.2)
IMGENEX/IMG-664AF488 FACS Ab
Anti-mouse CD45 FITC mAb (clone 30-F11) eBioscience
Anti-mouse F4/80 antigen PE mAb (clone BM8) eBioscience
Anti-mouse CD86 (B7-2) APC mAb (clone GL1) eBioscience
Anti-mouse CD11b FITC mAb (clone M1/70) eBioscience
Anti-mouse F4/80 antigen PerCP-Cyanine5.5 mAb (clone BM8)
eBioscience
Anti-mouse CD3 APC mAb (clone 17A2) eBioscience
Anti-mouse CD4 FITC mAb (clone GK1.5) eBioscience
Anti-mouse CD8a FITC mAb (clone 53-6.7) eBioscience
Anti-mouse CD11c PE mAb (clone N418) eBioscience
Anti-mouse Ly-6G (Gr-1) PE mAb (clone RB6-8C5) eBioscience
APC anti-mouse CD206 (MMR) mAb (clone C068C2) BioLegend
PE anti-mouse CD68 mAb (clone FA-11) BioLegend
APC anti-mouse Ly-6C mAb (clone HK1.4) BioLegend