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: jjmin@jnu.ac.kr (J.-J.M.); jhrhee@chonnam.ac.kr (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

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).

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

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,

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%

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).

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).

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.

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).

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).

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.

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).

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.

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,

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).

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).

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.

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.

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.

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).

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.

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.

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