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. 2023 Nov 2;31:100745. doi: 10.1016/j.omto.2023.100745

Bacterial cancer therapy using the attenuated fowl-adapted Salmonella enterica serovar Gallinarum

Daejin Lim 1, Kwangsoo Kim 2,3, Taner Duysak 3, EunA So 3, Jae-Ho Jeong 3,∗, Hyon E Choy 2,3,∗∗
PMCID: PMC10694566  PMID: 38053546

Abstract

We report here a novel anti-cancer therapy based on an avian-host-specific serotype Salmonella enterica serovar Gallinarum (S. Gallinarum) deficient in ppGpp synthesis. To monitor the tumor targeting, a bioluminescent ΔppGpp S. Gallinarum was constructed and injected intravenously into mice bearing syngeneic and human xenograft tumors. Strong bioluminescent signals were detected specifically in all grafted tumors at 2 days post-injection (dpi). The bacterial counts in normal and tumor tissue at 1 dpi revealed that ΔppGpp S. Gallinarum reached >108 CFU/g in tumor tissue and 106–107 CFU/g in endothelial organs; counts were much lower in other organs. At 16 dpi, ΔppGpp S. Gallinarum counts in tumor tissue decreased to ∼106 CFU/g, while those in the other organs became undetectable. A strong anti-cancer effect was observed after the injection of ΔppGpp S. Gallinarum into BALB/c mice grafted with CT26 colon cancer cells. This could be attributed to reduced virulence, which allowed the administration of at least a 10-fold greater dose (108 CFU) of ΔppGpp S. Gallinarum than other attenuated strains of S. enterica serovar Typhimurium (≤107 CFU). An advantage of the avian-specific S. Gallinarum as a cancer therapeutic should be a reduced capacity to cause infections or harm in humans.

Keywords: Salmonella Gallinarum, anti-cancer effect, mouse model, ppGpp

Graphical abstract

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Choy and colleagues developed a novel tool for anti-cancer therapy using an engineered strain based on fowl-infecting Salmonella enterica serovar Gallinarum, which demonstrates a distinct advantage in terms of reduced potential to harm humans during cancer treatment.

Introduction

It has been shown that live bacteria selectively accumulate and replicate in tumors and destroy cancer cells in animal models. Thus, the use of live bacteria for cancer therapy has recently attracted much attention. Accumulated bacteria initiate a series of immune-mediated anti-tumor responses, although the underlying mechanism remains unclear.1,2,3,4 Different variants of the genera Bifidobacterium, Clostridium, Lactococcus, Shigella, Vibrio, Listeria, Escherichia, and Salmonella have been analyzed in animal models of cancer.5,6,7,8,9 Among these, the facultative anaerobe Salmonella enterica serovar Typhimurium has been used most widely in experimental rodent models because of its amenability to genetic manipulation.10,11,12,13,14,15 However, rodents are highly susceptible to S. Typhimurium infection, and thus the bacteria need to be attenuated.

Among the many S. Typhimurium mutants that have been evaluated, VNP20009, A1-R, and ΔppGpp are the most widely studied.3 VNP20009 is a genetically modified auxotrophic strain with 108 kb deletion, purI−, msbB− , and many SNPs.16 The mutation in msbB gene resulted in a defective lipidA that crippled the associated virulence. Strain A1-R was chemically engineered to be auxotrophic for leucine and arginine,17 whereas ΔppGpp lacks a signaling molecule (ppGpp) that positively or negatively regulates gene transcription.18 In S. Typhimurium, ppGpp regulates transcription of all virulence genes encoded by Salmonella pathogenicity islands19,20; thus, the mutant defective in ppGpp synthesis, catalyzed by the relA and spoT gene products, was attenuated by over one million-fold compared with the wild-type strain.21 ΔppGpp S. Typhimurium strains carrying various anti-cancer protein drugs have been constructed and deployed to treat xenografted cancers in rodent models.22,23,24

S. enterica serotypes are divided into two groups on the basis of the disease caused and host tropism.25 Those with broad tropism include serovars Typhimurium and Enteritidis, which can cause enteritis in a wide range of host species; these two serotypes are responsible for the majority of food-borne Salmonella-induced enteritis cases in humans. Restricted host range serotypes rarely cause disease outside of their natural host, in which they cause systemic typhoid-like disease. Examples of these serotypes include S. Typhi in humans and S. Pullorum and S. Gallinarum in the chicken, which cause pullorum disease and fowl typhoid, respectively.26,27These diseases are attributed to the characteristics of Gallinarum/Pullorum serotype, which is incapable of entering the follicle-associated epithelium of mammalian Peyer’s patches; therefore, Gallinarum/Pullorum is unable to survive and multiply in the cells of the mammalian reticuloendothelial system in vivo and in vitro.28,29,30,31

Here, we constructed a novel anti-cancer strain on the basis of S. Gallinarum. We reported previously that a ppGpp-defective mutant of S. Gallinarum was highly attenuated in a white leghorn chicken model32 and that it was internalized by rodent-derived cells far less efficiently than wild-type bacteria; also, it failed to replicate intracellularly in vitro.31 We tested the ppGpp-defective S. Gallinarum in rodent cancer models and demonstrated a strong anti-cancer effect with few side effects.

Results

In an attempt to deploy S. Gallinarum as a cancer therapy agent, we first examined the survival of BALB/c mice injected intravenously (i.v.) with wild-type bacteria. We found that although S. Gallinarum causes typhoid only in fouls, all mice injected with >105 CFU died (Figure 1A). Therefore, we created a ppGpp-defective mutant by disrupting ppGpp synthetase I and II, encoded by relA and spoT genes, respectively.18 It has been reported that a defect in ppGpp confers S. Typhimurium and Escherichia coli auxotrophic for several amino acids. The ppGpp-defective S. Gallinarum also required several amino acids, including branched chain amino acids, lysine, and serine, to grow (Table S1).

Figure 1.

Figure 1

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Mice survival following injections of wild-type and ΔppGpp S. Gallinarum

Survival of BALB/c mice (n = 5 per group) after intravenous injection of wild-type (A; SG3021) or ΔppGpp S. Gallinarum (B; SG2023) at the indicated dosages (∗; < 0.025 and ∗∗∗∗p < 0.0001).

We then examined the tumor-targeting ability of the ppGpp-defective S. Gallinarum in tumor-bearing mice. To do this, we used bioluminescent bacteria and a noninvasive in vivo imaging system (IVIS). Bioluminescent bacteria were generated by transforming bacteria with an expression plasmid containing the luxCDABE operon from Photobacterium leiognathi.33 One major obstacle is that bacteria tend to lose the plasmid carrying the reporter gene, which is not needed for bacterial survival. In addition, it is impractical to use an antibiotic resistance gene as a selective determinant in animal experiment. Therefore, we used a balanced-lethal host-vector system (the glmS gene system) in S. Gallinarum. This system takes an advantage of the phenotype of the GlmS− mutant, which undergoes lysis in animals lacking the nutrients required for proliferation of the mutant (i.e., D-glucosamine [GlcN] or N-acetyl-D-glucosamine [GlcNAc], both of which are components of peptidoglycan synthesis).34,35,36 A novel balanced-lethal host-vector system was constructed in which the glmS gene of S. Gallinarum was incorporated into a plasmid that would complement the chromosomal glmS mutation. A 1.8 kb DNA fragment carrying the glmS of S. Gallinarum was obtained by PCR amplification and cloned into pLux containing the lux operon,33,37 thereby generating GlmS+pLux (Figure 2A). Luminescent signals from the bacteria carrying this plasmid that would be readily visualized by IVIS. We confirmed that the GlmS− mutant S. Gallinarum strain could not grow in ordinary bacterial growth medium unless supplemented with GlcNAc or GlcN and that the mutant was complemented with the GlmS+pLux.

Figure 2.

Figure 2

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The balanced-lethal host-vector system (glmS gene system) in S. Gallinarum

(A) Plasmid map of GlmS+pLux. (B) The GlmS− mutant strain (SG3031) or the parental strain (SG3023) carrying GlmS+pLux was sub-cultured (1:1,000) in LB broth every 12 h. The fraction of bacteria carrying the plasmid at the indicated time was determined by culture on GlcNAc-supplemented LB plates containing ampicillin (50 mg/mL). Each graph is representative of three independent experiments. Data represent mean ± SD, and asterisks indicate a significant difference between the wild-type and the GlmS− mutant (∗∗∗∗p < 0.0001).

The ppGpp-defective strain carrying either a wild-type or mutated glmS was transformed with GlmS+pLux carrying an ampicillin resistance marker, grown in Luria-Bertani (LB) broth, and sub-cultured (1:1,000) every 12 h. Bacteria carrying the plasmid were counted on GlcNAc-supplemented LB plates containing ampicillin. The results revealed that 99% of the plasmid carried by wild-type bacteria was cast out by day 4, while plasmids carried by the GlmS− mutant were fully maintained (Figure 2B). Therefore, the ppGpp-defective strain carrying a mutated glmS gene and transformed with GlmS+pLux was used for bioluminescence imaging.

Tumor targeting by ΔppGpp S. Gallinarum

Various rodent tumor models were generated by grafting cultured tumor cells into the right thigh of different mouse strains. BALB/c mice received CT26 colon cancer cells or mouse 4T1 breast cancer cells, C57/BL6 mice received B16F10 melanoma cells, and BALB/c athymic nu−/nu− mice received human AsPC-1 pancreatic cancer cells. When the grafted tumors reached ∼0.5 cm3 (after about 14 days), ΔppGpp S. Gallinarum harboring the mutation in glmS (1 × 108 CFU) and carrying GlmS+pLux were injected i.v. Bioluminescent signals generated by the bacteria were imaged at on the indicated days using an IVIS100 (Caliper, Hopkinton, MA) equipped with a cooled charge-coupled detector (CCD) camera (Figure 3). Following bacterial injection (∼20 min, 0 days post-injection [dpi]), bioluminescent signals were detected mainly in the reticuloendothelial system, namely, the liver and spleen. The bioluminescent signals from 2 dpi were detected exclusively in grafted tumor tissues; these signals diminished gradually over time. The signal generated in AsPC-1 peaked at 4 dpi. It should be noted that S. Gallinarum targeted all four different tumor grafts in mice used in this study. We also injected ΔppGpp S. Gallinarum carrying the wild-type glmS gene and the GlmS+pLux into BALB/c mice bearing CT26 tumors and tumor-associated bacteria were counted at 7 dpi using media plates containing ampicillin (Figure S1). The results showed that >90% of bacteria excreted the plasmid, demonstrating the necessity of using the balanced-lethal host-vector system. The presence of ΔppGpp S. Gallinarum in the grafted tumor tissue was demonstrated by staining bacteria in CT26 tumors at 3 dpi: bacteria were detected at the border between the proliferative and necrotic regions, as shown previously for E. coli21 (Figure 4).

Figure 3.

Figure 3

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Tumor-targeting ability of S. Gallinarum (SG2031) transformed with GlmS+pLux

The bacteria were administered intravenously to mice (n = 3 per group) grafted with tumors. Bioluminescence signals were measured at the indicated days post-infection (dpi) using IVIS100 equipped with a cooled charge-coupled detector (CCD) camera. The y axis indicates photons × 105 · S−1 · cm−2 · sr−1. Images labeled “0 dpi” were taken 20 min after bacterial injection.

Figure 4.

Figure 4

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Bacterial colonization in tumor tissue

The subcutaneous CT26 tumor tissues shown in Figure 3 were excised at 3 dpi and processed prior to staining with DAPI (nuclei), Alexa Fluor 488-conjugated phalloidin (actin), and a Salmonella-specific antibody. The confocal images are shown at 200× magnification. N, necrotic region; P, proliferative region.

Anti-cancer efficacy of ΔppGpp S. Gallinarum in the CT26 tumor mouse model

The in vivo anti-tumor activity of ΔppGpp S. Gallinarum was accessed by injecting 1 × 108 ΔppGpp S. Gallinarum into BALB/c mice bearing CT26 colon tumors. As a control, we also injected some mice with the ΔppGpp S. Typhimurium (1 × 107) used routinely in this laboratory.22,23,24,38,39 Tumors in mice treated with 1 × 108 ΔppGpp S. Gallinarum were significantly smaller than those in mice injected with 1 × 107 S. Typhimurium, although the latter retarded growth better than PBS (negative control) (Figures 5A and 5B). Consequently, the group treated with ΔppGpp S. Gallinarum survived for longer than the groups treated with ΔppGpp S. Typhimurium or PBS (Figure 5C). The median overall survival of mice treated with 1 × 107 S. Typhimurium was ∼24 days, while that of the mice treated with 1 × 108 S. Gallinarum was ∼53 days. Compared with the PBS control (∼15 days), treatment with 1 × 108 S. Gallinarum extended median overall survival by ∼3.5-fold. Under these conditions, we counted the actual number of bacteria in the internal organs and tumor tissue (Figure 6). At 1 dpi, the highest bacterial load was observed in the tumor (108–109 CFU/g), although this decreased gradually to ∼106 CFU/g. This was consistent with the observation that bioluminescence generated by S. Gallinarum carrying GlmS+pLux in the grafted tumor declined gradually over time (see Figure 3). The number of S. Gallinarum in the liver, spleen, and bone at 1 dpi was between 106 and 107 CFU/g; that in lung, kidney, and heart was ∼104 CFU/g; and that in the serum and eye was 101–103 CFU/g. Most notably, S. Gallinarum was cleared completely from these organs by 16 dpi, presumably because of host immune responses. At this time, the ratio of S. Gallinarum between the tumor and the reticuloendothelial system, spleen, liver, and other organs by 16 dpi would be infinite. Roughly the same pattern of ΔppGpp S. Typhimurium distribution was observed in these organs at 1 dpi (Figure S2). The number of S. Typhimurium in these organs, however, did not decrease much over the 16 days.

Figure 5.

Figure 5

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Anti-tumor effects of ΔppGpp S. Gallinarum (1 × 108 CFU, SG3023) compared with ΔppGpp S. Typhimurium (1 × 107 CFU, SMR2130) injected intravenously into BALB/c mice (n = 5/group) mice bearing CT26 colon tumors

(A) Average tumor volumes; error bars correspond to the 95% confidence interval (CI). Asterisks indicate significant differences from the PBS-treated control (∗p < 0.05 and ∗∗p < 0.005). (B) Representative gross morphological changes. (C) Tumor volumes in the individual mouse. (D) Kaplan-Meier survival curves for tumor-bearing mice receiving the treatments described above. ∗p < 0.03 and ∗∗p < 0.005.

Figure 6.

Figure 6

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Distribution of bacteria in organs on the indicated days post-intravenous injection of 1 × 108 Δ ppGpp S. Gallinarum (SG3022) into BALB/c mice (n = 5/group) carrying CT26 tumors

Data represent mean ± SD.

Discussion

In this study, we deployed 1 × 108 ΔppGpp S. Gallinarum into the various tumor-bearing mice through the i.v. route (Figures 1B and 3). Mice tolerated injection of up to 108 CFU easily. This is higher than the dose of ppGpp-defective S. Typhimurium used in our laboratory (∼107 CFU).22,23,24,39 Similar experiments with S. Typhimurium A1-R, which is capable of selectively infecting and attacking viable tumor tissue,40 as well as experiments with the purI−, msbB− mutant (VN20009)41,42 and the LPS-defective mutant ΔrfaG/ΔrfaD,38 revealed that mice tolerated a lower dose (106–107 CFU) of these bacteria. Tumor-bearing mice tolerated a dose of 108 CFU E. coli.41,43 Assuming that E. coli is almost completely avirulent, 108 CFU may be the most tolerable dose that does not trigger nonspecific catastrophe.

Endotoxemia induced by lipopolysaccharides (LPS) is a significant concern when using gram-negative bacteria for cancer therapy. In our case, especially because we used 1 × 108 S. Gallinarum, the endotoxemia is perceivable concern. A single E. coli cell is known to contain 2 × 106  molecules, which corresponds to an amount of about 20 fg.44,45 Assuming LPS in E. coli and S. Gallinarum are about the same, the LPS content in the 1 × 108 S. Gallinarum should be ∼2 μg. In general, the lethal dose of LPS typically falls within the range of 100 μg or more per mouse.46,47 Therefore, 2 μg of LPS in the 1 × 108 S. Gallinarum would be negligible. However, the lethal dose of LPS required to induce endotoxemia in different animals vary enormously depending on various factors, including the species, age, and health status, as well as the specific LPS source and route of administration.48

Bacteria-mediated cancer therapy relies on its ability to induce a systemic anti-tumor immune response.1,2,3,4,11,49 We observed an abrogated anti-cancer effect mediated by S. Gallinarum in the tumor-bearing mice depleted of either cytotoxic T lymphocytes (CD8+ T cells) or natural killer (NK) cells (Figure S3).The mechanism underlying the anti-tumor effect of S. Gallinarum would be much the same as that mediated by S. Typhimurium, in which cytotoxic CD8+ T cells or NK cells play pivotal roles.50,51 It is not yet certain, however, whether the striking anti-tumor effects observed with ppGpp-defective S. Gallinarum can be ascribed to reduced virulence, which allows the administration of a 10-fold greater dose than for ppGpp-defective S. Typhimurium, or to the intrinsic anti-tumor characteristics of S. Gallinarum. One factor clearly attributable to the reduced virulence of ppGpp-defective S. Gallinarum is rapid clearance from internal organs. S. Gallinarum appears to have several advantages over other strains as a cancer therapeutic: (1) it is safer than S. Typhimurium in humans and rodents, (2) it poses no health threat to the persons handling it in laboratories and clinics, and (3) using the balanced-lethal host-vector system means that the plasmids encoding anti-tumor protein-encoding genes are stably maintained.

Materials and methods

Bacterial growth

Salmonella spp were grown in LB broth (Difco Laboratories) containing 1% NaCl, with vigorous aeration at 37°C. For solid support, 1.5% granulated agar (Difco Laboratories) was included. Antibiotics were obtained from Sigma-Aldrich. When necessary, antibiotics were added at the following concentrations: ampicillin, 50 μg/m; kanamycin, 50 μg/mL; and chloramphenicol, 15 μg/mL. GlcNAc was added (100 mg/mL) to the medium as required.

Bacterial strains

The S. Gallinarum mutants were derived from clinical isolate SG3021 obtained from the liver of a chicken with fowl typhoid on a South Korean broiler farm (Table 1). All bacterial strains were constructed using the method developed by Datsenko and Wanner.52 The ppGpp-defective mutants were generated by sequential introduction of relA::kan and spoT::cat into the genome of SG3021. The open reading frame was replaced by a gene carrying either kan or cat by PCR amplification with a pair of 60 nt primers that include 40 nt homology extensions and 20 nt priming sequences; pKD13 was used as a template (Table 2). The PCR products were purified using DNA cleanup kit (#28104; Qiagen) and electroporated into bacteria carrying a lambda Red helper plasmid (pKD46). The mutants were confirmed by PCR using the original and test primers (Table 2). SG3023 was derived from SG3022 after eliminating antibiotic resistance genes using a helper plasmid expressing the FLP recombinase, pCP20. The GlmS− deletion mutant was generated in the same way.

Table 1.

Bacterial strains

Strain Description Reference or source
S. Gallinarum
SG3021 wild-type clinical isolate Jeong et al.31
SG3022 Δ relA::kan, Δ spoT::cat This work
SG3023 Δ relA,ΔspoT This work
SG3030 Δ relA,ΔspoT, glmS::kan This work
SG3031 ΔrelA,ΔspoT, ΔglmS This work
S. Typhimurium
SHJ2037 relA::kan, spoT::cat Song et al.19
SMR2130 Δ relA,ΔspoT Song et al.19
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Table 2.

Primers used

Primer name Sequence 5′-3′
relA::kan F GTG GAT CGC AAG CCT GGG AAT TTC CAG CCA GCA GTC GTG TGA GCG CTT AGG TGT AGG CTG GAG CTG CTT C
relA::kan R GTG CAG TCG CCG TGC ATC AAT CAC ATC CGG CAC CTG GTT CAG CTT ACC GAA TTC CGG GGA TCC GTC GAC C
spoT::cat F TTA AGC GTC TTC GGC AGG CGT ATC TCG TTG CAC GTG ACG CTC ACG AGG GCT GTA GGC TGG AGC TGC TTC
spoT::cat R GCC AGA TGT ACG CGA TCG CGT GCG GTA AGG CGA ATA AAG GTA CTA TAG ACC ATA TGA ATA TCC TTA G
glmS::kan F TTA CTC AAC CGT AAC CGA TTT TGC CAG GTT ACG CGG CTG GTC AAC GTC GGT GCC TTG ATT GTG TAG GCT GGA GCT TCG AA
glmS::kan R ATG TGT GGA ATT GTT GGC GCG ATC GCG CTT CGT GAT GTA GCT GAA TCC TTC TTG AAG GTC ATA TGA ATA TCC TTC GTT CC
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Measurement of plasmid stability

Overnight cultures were sub-cultured (1:1,000) every 12 h in fresh LB medium. Samples were taken every 24 h and serially diluted. Appropriate volumes were spread in triplicate on GlcNAc-supplemented LB plates with or without ampicillin. The number of colonies was used to calculate the total number of viable cells (CFU) and the fraction of bacteria containing the plasmid.

Plasmid GlmS+pLux

The luminescence-expressing plasmid (pLux) was described previously.33 Briefly, pLux containing the lux operon (luxCDABE, ∼9.5 kb) of P. leiognathi was inserted into the pUC19 plasmid backbone via the XbaI restriction enzyme sites.37 To construct a plasmid containing both the lux operon cassette and glmS, the glmS gene of S. Gallinarum was amplified using forward primer 5′-AA GTC GAC ATG TGT GGA ATT GTT GGC-3′ and reverse primer 5-GG GTC GAC TTA CTC TAC GGT AAC CGA TTT C-3′, with genomic DNA as a template. This 1.8 kb fragment was digested with Sal I and ligated into the same site in the pLux vector to generate GlmS+pLux (Figure 2A).

Tumor cell lines

Murine CT26 colon carcinoma cells, 4T1 breast carcinoma cells, B16F10 melanoma cells, and human ASPC1 pancreas adenocarcinoma cells were obtained from the American Type Culture Collection. The CT26 and 4T1cells were grown in high-glucose DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin. The murine B16F10 melanoma cells and ASPC1 cells were grown in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin.

Bacterial distribution in internal organs in rodents

For the viable counts of Salmonella spp, groups of BALB/C mice (n = 5) were injected with 1 × 107 ΔppGpp S. typhimurium and 1 × 108 ΔppGpp S. Gallinarum through the i.v. route and sacrificed on the days specified (day 1, 5, 10, or 16). The organs were collected and the tissues were homogenized in sterile PBS containing 0.05% Tween 20 with homogenizer. The Salmonella was recovered from the homogenates and quantified by plating on agar plates containing kanamycin (50 μg/mL) and chloramphenicol (15 μg/mL).

Experiments using rodents as cancer models

Male mice (5–6 weeks old, 20–30 g body weight) were purchased from Samtako Company. All animal care, experimental, and euthanasia procedures were performed in accordance with approved protocols. Mice carrying subcutaneous tumors were generated as follows: tumor cells cultured in vitro were harvested, suspended in 30 μL PBS, and injected subcutaneously into the right thigh at the indicated doses (1 × 106 cells for CT26, 4T-1 and B16F10 cells; and 1 × 107 cells for ASPC1). When tumors reached ∼100 mm3, ppGpp-defective S. Gallinarum or S. Typhimurium (1 × 108 and 1 × 107 CFU/mL in PBS, respectively) were administrated through the tail vain (day 0). The tumor volume was calculated using the following formula: (L × H × W)/2, where L is the length, W is the width, and H is the height of the tumor in millimeters. Mice with tumor volumes ≥1,500 mm3 were euthanized according to the guidelines of the Animal Research Committee. Survival rates were estimated using the Gehan-Breslow-Wilcoxon test. All animal experiments were approved and performed by the Institutional Animal Use and Care Committee of Chonnam National University (CNU IACUC-H-2020-7).

Optical bioluminescence imaging

To image bacterial bioluminescence, animals were anesthetized with isoflurane (2%)24 and placed in the light-tight chamber of the IVIS100, which was equipped with a cooled CCD camera. Photons emitted from luciferase-expressing bacteria were collected and integrated over 1 min periods. Pseudocolor images indicating photon counts were overlaid on photographs of the mice using the Living Image software version 2.25 (Caliper).

Immunofluorescence staining and confocal microscopy

To stain Salmonella inside the tumor, isolated tissues were fixed overnight at room temperature in PBS containing 3.4% formalin and embedded in optimal cutting temperature (OCT) compound (Tissue-Tek). The tissues were then frozen and sliced into 7 mm sections using a microtome-cryostat. The sections were collected on aminopropyl triethoxysilane-coated slides, washed with PBS (pH 7.4) to remove the OCT, and incubated overnight at 4°C with an anti-Salmonella LPS antibody (1:100; #ab8274; Abcam), followed by an Alexa Fluor 568-conjugated goat anti-mouse (1:100; #A11031; Life Technologies) and Alexa Fluor 488-conjugated phalloidin (1:1,000; #W21404; Invitrogen) secondary antibodies and for the detection of F-actin (1:1,000, #MA1-80729; Thermo Fisher Scientific), respectively. After staining nuclei with DAPI/Antifade (1:200; Invitrogen), the samples were mounted and images were captured under a confocal microscope (LSM 510; Zeiss Laboratories). Representative images are shown in the figures.

Statistical analyses

Statistical analysis was performed using SPSS 18.0 (SPSS Inc., Chicago, IL). A two-tailed Student’s t test was used to determine the significance of differences in tumor growth between the control and treatment groups. A p value of 0.05 was considered to indicate statistical significance. All data are expressed as mean ± SD.

Data and code availability

All data generated or analyzed in this article are available from the corresponding author upon request.

Acknowledgments

This work was supported by the Starting Growth Technological R&D Program (TIPS Program; grant S3130592) funded by the Ministry of SMEs and Startups (MSS; Korea) in 2021. J.-H.J. was supported by a National Research Foundation of Korea (NRF) grant (MSIT) (2020R1A5A2031185). D.L. was supported by a 2023 Research Grant from Kangwon National University and a National Research Foundation of Korea grant funded by the Korean government (MSIP) (NRF-RS-2023-00210053).

Author contributions

Conceptualization, H.E.C. and J.-H.J.; methodology, K.K., T.D., E.S., and D.L.; investigation, E.S., T.D., K.K., and D.L.; writing – original draft, D.L.; writing – review & editing: H.E.C., J.-H.J., and D.L. All authors have read and agreed to the published version of the manuscript.

Declaration of interests

The authors declare no conflicts of interest.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.omto.2023.100745.

Contributor Information

Jae-Ho Jeong, Email: jeongjaeho@jnu.ac.kr.

Hyon E. Choy, Email: hyonchoy@chonnam.ac.kr.

Supplemental information

Document S1. Figures S1–S3 and Table S1
mmc1.pdf (583.5KB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (3.9MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S3 and Table S1
mmc1.pdf (583.5KB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (3.9MB, pdf)

Data Availability Statement

All data generated or analyzed in this article are available from the corresponding author upon request.

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