A polymer coating applied to Salmonella prevents the binding of Salmonella-specific antibodies
Che-Hsin Lee1,2＊, Yu-Hsin Lin3, Jeng-Long Hsieh4, Man-Chin Chen2 and Wan-Lin
Kuo1
1Department of Microbiology, School of Medicine, China Medical University,
Taichung, Taiwan
2Graduate Institute of Basic Medical Science, School of Medicine, China Medical
University, Taichung, Taiwan
3Department of Biological Science and Technology, China Medical University,
Taichung, Taiwan
4Department of Nursing, Chung Hwa University of Medical Technology, Tainan,
Taiwan
Address correspondence and reprint requests to: Dr. Che-Hsin Lee, Department of Microbiology, School of Medicine, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan. E-mail: [email protected]; Fax: +886-422053764; Tel: +886-4-22053366 ext:2173
Keywords: Salmonella; poly(allylamine hydrochloride); neutralizing antibody; tumor-targeting
Journal category: Cancer Therapy
Salmonella following in vivo delivery. The polymer-modified Salmonella not only
resist the biding of the neutralizing antibodies in peripheral blood but also replicate in tumor sites, which are believed to stimulate non-specific antitumor immunity. These results help define the obstacles necessary to overcome for a successful bacteriolytic therapy.
Abstract
The use of Salmonella as a potential antitumor agent has been investigated, but innate immunity against this bacterium reduces the efficacy of its tumor-targeting and antitumor activities. The purpose of this study was to investigate the modulation of the tumor-targeting efficiency of Salmonella enterica serovar choleraesuis by modifying the immune response to these bacteria by coating them with poly(allylamine hydrochloride) (PAH), designated PAH-S.C. To evaluate this modulation, we used naïve mice and mice immunized with Salmonella to study the role of the preexisting immune response to the antitumor activity of PAH-S.C. When anti-Salmonella antibodies were present, the invasion activity, cytotoxicity, and gene transfer of Salmonella was significantly decreased, both in vitro and in vivo.
Treatment with PAH-S.C. resulted in delayed tumor growth and enhanced survival in immunized mice. Furthermore, immunohistochemical studies of the tumors revealed the infiltration of neutrophils and macrophages in immunized mice treated with PAH-S.C. These results indicate that Salmonella encapsulation effectively circumvented the Salmonella-specific immune response.
Introduction
One of the primary limitations of cancer therapy is the lack of selectivity of
therapeutic agents against tumor cells. The use of preferentially replicating bacteria as an oncolytic agent is an innovative approach for cancer treatment.1-3 This approach is
based on the observation that several obligate or facultative anaerobic bacteria are capable of selectively multiplying in tumors and thereby inhibiting their growth.4
Salmonella are gram-negative, facultative anaerobes that are a common cause of
intestinal infections. Salmonella grow under aerobic and anaerobic conditions; thus, they are able to colonize small metastatic and larger tumors. Attenuated Salmonella have been shown to inhibit tumor growth in a broad range of human and mouse tumors.5
Salmonella-based vectors have been considered as potential antitumor agents for
tumor vaccines, gene delivery and tumor-targeting vectors.6-8 However, a major
disadvantage to this approach is the development of immunity to Salmonella.9
Salmonella-specific antibodies significantly reduce the tumor-targeting activity of Salmonella following a second administration of Salmonella. Humoral responses,
particularly those involved in the production of neutralizing antibodies, reduce
Salmonella’s antitumor activity. Moreover, preexisting Salmonella neutralizing
activity even at the first inoculation of Salmonella. Masking Salmonella
immunogenicity with a linking polymer is one approach that can be used to inhibit antibody-mediated Salmonella neutralization.
Purified proteins, plasmids, and viruses can be encapsulated in a biodegradable polymer and delivered by various routes.11 The interactions of polymers with
biological systems has been a topic of interest in widely divergent fields. Living cells can be encapsulated by the alternating adsorption of oppositely charged
polyelectrolytes, and the metabolic activity of the coated cells is well preserved after encapsulation.12-13 The bacterial cell wall is negatively charged due to the presence of
either teichoic acid in positive bacteria or lipopolysaccharide (LPS) in gram-negative bacteria. Positively charged polyelectrolytes can bond to gram-negatively charged bacterial surfaces to form thin films that circumvent preexisting immunity against bacterial vectors. In addition to rendering tumor-targeting Salmonella less susceptible to inactivation by neutralizing antibodies, this study demonstrates that the masking of
Salmonella with a polymer reduces the antigenicity of Salmonella following in vivo
Materials and Methods
Bacteria, cell lines, reagents and mice
The Salmonella enterica serovar choleraesuis (S. Choleraesuis; S.C.) (ATCC 15480) vaccine strain was obtained from the Bioresources Collection and Research Center (Hsinchu, Taiwan). This rough variant of S.C., which is designated vaccine 51, was obtained by spreading an 18-h broth culture of the virulent strain 188 of the S. Choleraesuis serovar Dublin over the surface of a dried nutrient agar plate, adding a drop of a suspension of salmonella anti-o phage No. 1, and selecting for a phage-resistant colony after incubation at 37ºC for 24 h.8-9 S.C. were transformed with
pTCYLuc by electroporation to generate S.C./Luc as previously described.4 Murine
4T1 breast tumor cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 50 μg/ml gentamicin, 2 mML-glutamine and 10% heat-inactivated fetal bovine serum at 37ºC in 5% CO2.14 Poly(allylamine hydrochloride)
(PAH; MW: 15,000), 4’,6-diamidino-2-phenylindole (DAPI), fluorescein amine isomer I (FA), and paraformaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). Six-to-eight-week-old female BABL/c mice were obtained from the National Laboratory Animal Center of Taiwan. The animals were maintained in a specialized pathogen-free animal care facility in isothermal conditions with regular photoperiods. The experimental protocol adhered to the rules of the Animal Protection
of the China Medical University (permit number: 99-20-N).
Preparation of PAH-modified Salmonella
S.C. (106 colony-forming units (cfu)) were washed with deionized water to
remove nutrients and metabolites from the bacteria. The washed S.C. were dissolved in 1 ml of PAH solution (1.25 mg/ml - 20 mg/ml), incubated on a shaker for 15 min and then centrifuged (10 min at 1,000 g). The excess PAH solution was discarded, and the S.C. were dispersed and washed three times in water.
Characterization of the PAH-modified Salmonella
The size distributions and zeta potential values of the S.C. and the PAH-modified S.C. (PAH-S.C.) were measured in deionized water using a dynamic light scattering system (Zetasizer ZS90, Malvern instruments, Malvern, UK). The synthesis of FA was based on a reaction between the FA amine groups and the PAH carboxylic acid groups. PAH and FA were dissolved in acetonitrile and incubated at room
temperature for 2 h. To remove the unconjugated FA, the solution was dialyzed in the dark in distilled water, which was replaced daily until no fluorescence was detected in the supernatant. The resultant FA-PAH was lyophilized in a freeze dryer.15 The
PAH-S.C and FA-PAH-PAH-S.C. cells were prepared as previously described . The PAH-PAH-S.C. and S.C. incubated 4T1 cells, respectively, for 2 h. The cells were then washed with PBS, fixed in 3.7% paraformaldehyde and observed by confocal laser scanning microscopy. The images were superimposed using the LCS Lite software. The
FA-PAH-S.C. were collected at various time points before the measurements. The fluorescence emission from the solutions was measured using a fluorescence spectrometer (LS50B, PerkinElmer, Emeryville, CA, USA).
Animal Studies
Groups of mice were intraperitoneally (i.p.) immunized with heat-killed S.C. or PAH-S.C. at a dose of 104 cfu. The immunized and naïve mice were bled or sacrificed
at several time points to determine antibody production.9 Other groups of naïve and
immunized mice were subcutaneously (s.c.) inoculated with 106 tumor cells. When
the tumors had grown to diameters between 50and 100 mm3, the mice were
intravenously (i.v.) injected with 5 mg/ml PAH or 2 × 106 cfu of S.C. or PAH-S.C.
These groups of mice were sacrificed at various time points postinfection, and the numbers of Salmonella in the tumors, livers, and spleens were determined on LB agar plates; these data were expressed as cfu per gram of tissue. Tissue homogenates were also assessed for luciferase activity using a luciferase reporter gene assay system (Tropix). In a parallel experiment, the fluorescence emission from solutions was measured using a fluorescence plate reader (Tecan Safire II, Salzburg, Austria). In a separate experiment, palpable tumors were measured every 3 days in two
perpendicular axes using a tissue caliper, and the tumor volumes were calculated as follows: (length of tumor) × (width of tumor)2 × 0.45. The survival rates of the mice
in the treated and control groups were monitored daily.
Infection of tumor cells with Salmonella
S.C. (106 cfu) or PAH-S.C. (106 cfu) were incubated with various sera collected
from mice and cultured with 4T1 cells (105 cells/well) for 8 h. The medium was then
removed, the cells were washed, and fresh medium supplemented with 50 μg/ml gentamicin was added. The supernatants were removed after an additional 90 min culture ingentamicin, and adherent cells were lysed to release the intracellular bacteria. The lysates were seriallydiluted in PBS and spread on LB agar plates, and the cfu were countedafter an overnight incubation at 37°C. In a parallel experiment, the adherent cells were measured for cell survival. Cell survival was assessed using the colorimetric WST-1 assay (Dojindo Labs, Tokyo, Japan) according to the manufacturer’s instructions. Tumor cells (105/well) were cultured in 6-well plates
overnight. Subsequently, S.C./Luc or PAH-S.C./Luc admixed with various sera collected from mice were added to cells that were then cultured in 1 ml of antibiotic-free medium and incubated for 8 h. All the cells were washed, replenished with complete medium containing gentamicin (50 μg/ml) and cultured for 16 h. The cells were lysed to prepare extracts for the determination of luciferase activity using a luciferase assay kit (Tropix).
Assessment of cytokines and immunohistochemical staining
(IL-1β) in the sera of the mice after S.C. or PAH-S.C. administration were determined by enzyme-linked immunosorbent assay (ELISA) (R＆D, Minneapolis, MN, USA). To analyze cell infiltration in the tumors, groups of mice that had been inoculated s.c. with 106 4T1 cells at day 0 were injected i.v. with 2 × 106 cfu of S.C. or PAH-S.C. at
day 10, and the control mice received PBS. The tumors were excised and snap-frozen on day 20. Cryostat sections (5 μm) were prepared, fixed, and incubated with rat mouse Ly 6G (Gr-1) (RB6-8C5, BD Biosciences, San Diego, CA, USA) or rat anti-mouse Mac-3 (M3/84, BD Biosciences) antibodies. After sequential incubation with the appropriate peroxidase-labeled secondary antibody and aminoethyl carbazole (AEC) as the substrate chromogen, the slides were counterstained with hematoxylin. The numbers of infiltrating cells were quantified by averaging the number in each cell type in the three areas of highest cell density at 200X magnification in each section.15
Statistical Analysis
The unpaired, two-tailed Student’s t test was used to determine differences between groups for the comparison of tumor volume, Salmonella luciferase activity, cell number, body weight and cytokine production. A survival analysis was performed using the Kaplan-Meier survival curve and log-rank test. A p value less than 0.05 was considered to be statistically significant.
Results
Characterization of poly(allylamine hydrochloride) (PAH)-modified Salmonella
Because the surface charge of the bacterial wall is negative, the positively charged polyelectrolyte PAH is effectively and spontaneously absorbed onto the bacterial cell wall.13 The particle sizes and surface charges of PAH-S.C. cells prepared with
different PAH concentrations were measured; the results are listed in Table 1.
Dynamic light scattering analysis demonstrated that the average size of the PAH-S.C. particles increased with increasing PAH concentration. The surface charges of the S.C. and PAH-S.C. particles were measured by zeta potential. The net surface charges of the PAH-S.C. and S.C. particles ranged from -4.07 to 11.75 mV. The gradual increase in particle size and neutralization of the Salmonella surface charge suggest that the Salmonella were well coated with PAH.
We also measured the replication rates of PAH-S.C. and S.C. cells to determine whether the PAH coating on the surface of Salmonella affected the physiology of the bacterium. When Salmonella were coated with 20 mg/ml PAH, the Salmonella growth curve was slightly decreased compared with the control (Fig. 1a). The growth rates of the other groups were not significantly different from that of the S.C. group. At 20 mg/ml, PAH may affect the growth of bacteria by inhibiting cell wall formation.
To test whether the PAH-S.C. cells were able to infect tumor cells and to determine their infection efficiency in the absence and presence of a neutralizing
antibody, the gentamicin protection assay was used to measure the degree of infection of tumor cells by PAH-S.C. The bacterial invasion assay demonstrated that the invasion efficiency of PAH-S.C. decreased with increasing PAH concentration (Fig. 1b). The same phenomenon was observed in the presence of a naïve antibody.
Interestingly, the PAH-S.C. prepared with 5 mg/ml PAH retained invasion activity in the presence of a neutralizing antibody. The PAH-S.C. prepared with 20 mg/ml PAH lost invasion activity and cytotoxicity, both in the absence and presence of a
neutralizing antibody (Fig. 1c).
Previously, we demonstrated that Salmonella are effective gene transfer vectors in
vitro and in vivo. 4, 16 To test whether PAH-S.C. were able to transfer genes into tumor
cells, luciferase expression was measured after infection with PAH-S.C. carrying a luciferase gene (PAH-S.C./Luc) (Fig. 1d). Tumor cells infected with PAH-S.C. prepared with 20 mg/ml PAH displayed no significant luciferase signals, either in the absence or presence of a neutralizing antibody. Notably, the expression of luciferase in PAH-S.C. in the 5 mg/ml group was observed both in the absence and presence of a neutralizing antibody. Our results demonstrated that the anti-Salmonella antibody did not inhibit PAH-S.C. invasion. Therefore, coating Salmonella with 20 mg/ml PAH partially decreased its infection ability, and coating Salmonella with 1.25 mg/ml PAH did not protect Salmonella from the neutralizing antibody. The PAH-S.C. cells
prepared with 5 mg/ml PAH were therefore used in the subsequent analyses. To further demonstrate that PAH did indeed adhere to the Salmonella surface, PAH was fluorescently labeled with FA to form FA-PAH. As illustrated in Fig. 2, the
Salmonella cells were well covered with FA-PHA (Fig. 2a). We hypothesized that the Salmonella PAH coating would be diluted with each subsequent cell division.
Furthermore, to determine the kinetics of PAH release in vitro, we collected the bacteria and removed the supernatants at several time points. As expected, the level of PAH coating continuously decreased during bacterial division (Fig. 2b). Additionally, we measured the fluorescence of the tumor homogenate after FA-PAH-S.C.
administration. The FA-PAH levels in the Salmonella recovered from the tumors were determined at various time points (Fig. 2c). The fluorescence in the tumors 48 h after injecting the mice with FA-PAH-S.C. was approximately 20-fold lower than that in the tumors at 12 h. This is in contrast to the kinetics of bacterial accumulation in tumors. Our data support the hypothesis that the PAH-S.C. retained the ability to infect tumor cells in vitro and in vivo.
Tissue distributions of PAH-S.C. in naïve and immunized mice
To investigate the impact of immune responses on the tissue distribution of S.C., naïve and immunized mice bearing tumors were i.v. injected with PAH-S.C., and the amounts of Salmonella in the tumors, livers, and spleens were
determined at days 1 and 10. As shown in Fig. 3a, systemically administered S.C. or PAH-S.C. preferentially accumulated within the tumors on day 1, at tumor-to-normal tissue accumulation ratios of 1000-10000:1. The amounts of S.C. and PAH-S.C.in the tumors remained at high levels over the ten days (Fig. 3b). We previously obtained similar results demonstrating that the bacterial load in tumors was significantly reduced in mice immunized against Salmonella.9 These findings suggested that the
immune response inhibited the accumulation of Salmonella in the tumor sites. Notably, the PAH-S.C. restored the tumor-targeting ability of Salmonella in immunized mice.
As shown in Fig. 3b, ten days after PAH-S.C. inoculation, the amounts of
Salmonella in the tumors were approximately four orders of magnitude higher than
those found in tumors derived from immunized mice treated with S.C. In addition, luciferase expression was readily observed in the tumor sites after PAH-S.C./Luc inoculation (Fig. 3c). The PAH-S.C. displayed tumor-targeting efficiency and gene transfer ability in the presence of a host with anti-Salmonella immunity. Our data demonstrated that PAH-S.C. circumvented the anti-Salmonella antibodies in vitro and
in vivo.
Effects of PAH-modified Salmonella on cytokine induction in naïve mice
infection, the administration of agents such as TNF-α, TNF-α neutralizing antibodies, and IL-1 receptor antagonists may reduce the biological activity of these cytokines and their side effects.17 To examine the host inflammation response induced by
PAH-S.C., immunized and naïve mice were treated with S.C. or PAH-S.C, and body weight was measured. As indicated in Fig. 4a, naïve mice treated with S.C. had a 9% lower average body weight compared with naïve mice treated with PBS. In contrast, the body weights of mice treated with PAH-S.C. were not significantly decreased. After S.C. and PAH-S.C. treatment, the levels of inflammatory cytokines including IL-1β and TNF-α were measured in the sera. Regardless of whether the mice were naïve or immunized, the induction of inflammation cytokines (i.e., IL-1β and TNF-α) in mice treated with S.C. was increased by 1.5-4.5-fold compared with the induction by PAH-S.C. treatment (Fig4. b and c). Taken together, these results suggest that PAH-PAH-S.C. had a greater tumor-targeting efficiency with potentially fewer side effects in the host than S.C.
Inhibition of tumor growth by PAH-modified Salmonella
Previously, we demonstrated that the presence of anti-Salmonella antibodies results in a noticeably lower total number of bacteria in tumor sites and decreases the antitumor effect of Salmonella.9 Therefore, the avoidance of neutralizing antibodies
antitumor effects of PAH-S.C. and S.C. were evaluated in terms of tumor growth and survival in naïve and immunized mice bearing tumors. S.C. and PAH-S.C. treatment both reduced tumor growth compared with PBS treatment in naïve mice.
Interestingly, PAH-S.C. treatment significantly reduced tumor size compared with S.C. treatment in immunized mice (Fig. 5a). The mean tumor volume for immunized mice in the PAH-S.C.-treated group decreased by 48.72% compared with those in the S.C.-treated group. Fig. 5b demonstrates that the survival of the immunized mice injected with PAH-S.C. was significantly prolonged compared with that of the mice injected with S.C. To further confirm that PAH protects against neutralizing
antibodies, we used PAH-S.C. to immunize mice. The anti-PAH-S.C. antibodies did not inhibit the antitumor activity of S.C (Fig. 5c). Furthermore, PAH did not produce an antitumor response. These results are consistent with our previous report that demonstrated that the specificity of anti-Salmonella antibodies influences the tumor-targeting potential of Salmonella.9 Overall, PAH-S.C. significantly suppressed tumor
growth and prolonged the survival of immunized mice.
Increase of infiltrating cells by PAH-S.C.
The antitumor properties of Salmonella may partially rely on the activation of host immunity.18-21 Neutrophil and macrophage infiltration in tumors from
postinfection by immunohistochemistry. The results of the immunohistochemical staining are presented in Fig. 6a. A few macrophages and neutrophils were detected in the tumors derived from immunized mice treated with S.C. Nevertheless, a notable increase in macrophage and neutrophil infiltration in the tumors was observed in naïve mice treated with PAH-S.C. and, in particular, in immunized mice treated with PAH-S.C. The numbers of infiltrating neutrophils and macrophages in the tumors treated with S.C. and PAH-S.C. were significantly increased compared with those in tumors treated with PBS (Figs. 5b and c). Notably, the number of infiltrating cells in tumors derived from immunized mice treated with PAH-S.C. was significantly increased compared with those treated with S.C. Taken together, these results reveal that PAH-S.C. cells enhanced immune cell infiltration in tumors in immunized mice. PAH-S.C. not only resisted the binding of neutralizing antibodies in peripheral blood but also replicated in tumor sites, which has been suggested to stimulate non-specific antitumor immunity.
Discussion
Systemic administration of Salmonella in tumor-bearing mice leads to its preferential accumulation in tumor sites and thus retards tumor growth. Salmonella can effectively eradicate primary and metastatic tumors including bone, prostate, breast, pancreas and sarcoma.22-25 Many studies suggest the clinical potential of bacterial treatment for
critical metastatic tumor targets.26-29 Salmonella with an attenuated lipid A have been
evaluated in a Phase I clinical trial, in which patients received Salmonella
(VNP20009), which was rapidly cleared from the blood, and the majority of tumors had no detectable Salmonella colonization. Moreover, patients that had preexisting anti-Salmonella antibodies had no detectable Salmonella colonization in the tumor sites after systemic administration.17 In agreement with a clinical study, our previous
results indicated that higher anti-Salmonella antibody titers in the host resulted in lower amounts of Salmonella in tumor sites. Anti-Salmonella antibodies result in a noticeably lower total number of bacteria in tumor sites and decrease the antitumor effect of Salmonella. Therefore, the avoidance of neutralizing antibodies has emerged as a key issue in the development of Salmonella therapeutic approaches. In this study, we investigated the use of PAH to modify or shield Salmonella from the preexisting immune response in a host.
coating Salmonella with such a film would not seriously affect its viability or invasion ability. A high concentration of PAH (20 mg/ml) may alter the bacterial outer
membrane permeability by binding to the negatively charged LPS layer, resulting in a destabilized outer membrane that inhibits bacterial replication and invasion.
Conversely, a low PAH concentration (1.25mg/ml) was insufficient to block
Salmonella antigenicity and protect against antibody neutralization.
Because toxic adverse effects caused by bacterial therapy are the main hindrance to further clinical application,16 we explored the use of PAH-S.C., which did not
induce a large response of inflammatory cytokines; such an inflammatory response would negatively affect the health of the host. Compared with S.C., PAH-S.C. exhibited greater safety in all the tested parameters including bacterial accumulation in tissue, inflammatory cytokine induction, and weight loss.
Because bacterial replication in tumors and the subsequent lysis of tumor cells may induce cell-mediated immune responses to tumor cells, higher oncolysis could, in part, account for increased infiltration of CD8+ T-cells in Salmonella-treated tumors.6, 21 The T-cell response against tumor cells may enhance the antitumor efficacy of
Salmonella. Saccheri et al. observed an antimicrobial response present in tumors that
activates cytotoxic CD8+ T-cells, which can recognize and kill tumor cells.6 The use
mice after Salmonella administration. These results not only indicated that T-cell mechanisms are important for the control of systemic Salmonella treatment but further suggested that T-cells participate in antitumor activities directed against
Salmonella. We also found that CD8+ T cells are antitumor effectors of Salmonella.
Furthermore, Salmonella induced IFN-γ production and polarized the T-cell response to a Th1-dominant state in wild-type mice but not in CD4+ T-cell-deficient
mice. Thus, a bacterially activated CD4+ T-cell tumor infiltration may be a relevant
source of IFN-γ in the tumor microenvironment and may contribute, a least in part, to the host antitumor immunity induced by Salmonella. The current findings and our previous reports suggest that tumor-targeted therapy involving Salmonella, which exerts tumoricidal effects and stimulates T-cell activities, represents a potential strategy for tumor treatment.30 However, persistently high levels of PAH-S.C. in
tumors induced inflammatory responses, leading to the recruitment of immune cells including macrophages and neutrophils to the tumor site (Fig. 6). Hosts immunized with Salmonella can induce an antitumor response due to the ability of
anti-Salmonella immune cells to kill anti-Salmonella-infected tumor cells.31 The antitumor
effects of neutrophils, in particular, after being activated by substances derived from microorganisms have also been demonstrated in various tumors.32-34
cells display lower toxicity and improved efficacy and safety. PAH also can provide a useful platform for the chemical modification of Salmonella , perhaps allowing other chemotherapeutic drugs to bind to tumor-targeting Salmonella. By taking advantage of the tumor-targeting activity of Salmonella and the pleiotropic activities of
chemotherapeutic drugs, this system appears to hold promise for tumor treatment. These results yield insight into the complex interactions between Salmonella and host immunity, maximizing the possibility of therapeutic success. Therefore, PAH-S.C. has promising potential for further clinical studies.
Grant sponsor
National Science Council and China Medical University Grant number:
NSC 100-2320-B-039-024; NSC 100-2815-C-039-031-B; CMU99-N2-08 and CMU100-N2-01
Acknowledgements
The authors thank Dvaid L. for carefully proof-reading the manuscript and providing valuable comments.
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Table 1
Particle sizes and zeta potential values of PAH-S.C. particles prepared with different PAH concentrations in deionized water (n=5).
PAH concentration (mg/ml) Particle size (nm) Zeta potential (mV) 0 938.13 ± 84.69 -4.70 ± 2.45 1.25 1281.14 ± 71.61 -0.7469 ± 2.04 5 1180.57 ± 63.76 4.22 ± 1.18 20 1260.55 ± 180.33 11.75 ± 0.31
Figure legends
Figure 1. Replication, invasion activity, cytotoxic effect, and transduction ability of PAH-S.C. (a) PAH-S.C. replication. The number of PAH-S.C. cells was
determined 6 h postincubation. (b) The mice were immunized i.p. with 104 cfu of
heat-killed S.C. on days 0 and 7. The naïve mice were injected with PBS. The sera were collected 7 days after the last immunization. 4T1 cells (105) were infected with
106 cfu of PAH-S.C. or S.C. cells admixed with various sera obtained from
S.C.-immunized or naïve mice. A gentamicin protection assay was used to examine these cells 9.5 h later; the data are reported as means ± SD (n=3) (c) The effect of
neutralizing antibodies on cell death induced by Salmonella infection. 4T1 (105) cells
were infected with S.C. (106 cfu), S.C. admixed with various sera, PAH-S.C. (106
cfu), or PAH-S.C. admixed with various sera. The cell viability was then assessed using the WST-1 assay; the data are reported as means ± SD (n=6). (d) The effect of neutralizing antibodies on gene transfer in vitro. 4T1 (105) cells were infected with
S.C./Luc (106 cfu), PAH-S.C./Luc (106 cfu), S.C./Luc admixed with sera or
PAH-S.C./Luc admixed with sera. Luciferase activities in these cells were measured 16 h later. (*, p<0.05, ***, p<0.001).
Figure 2. Salmonella (S.C.) coated with PAH. (a) 4T1 cells treated with FA-PAH-S.C. were imaged by confocal microscopy. Cell nuclei and bacterial DNA were counterstained with DAPI. Scale bar = 8 μm. (b) Kinetics of PAH release. FA-PAH-S.C. cells were collected at several time points. The fluorescence emissions from the solutions were measured using a fluorescence spectrometer. The numbers of
Salmonella were determined at the various time points. (c) Mice bearing 4T1 tumors
ranging in size from 50 - 100 mm3 were injected i.v. with FA-PAH-S.C. (2 ×106 cfu)
at 10 days, and the fluorescence emissions from the tumor homogenates were measured using a fluorescence spectrometer. The numbers of Salmonella in the tumors were determined at several time points, and the data are reported as means ± SD (n=3).
Figure 3. The effects of neutralizing antibodies on the tumor-targeting potential and gene transfer of Salmonella (S.C.). The mice were immunized i.p. with 104 cfu
of heat-killed S.C., and the control mice were immunized with PBS. The mice were subcutaneously injected with tumor cells (106). Mice bearing 4T1 tumors ranging
from 50 - 100 mm3 were injected i.v. with S.C. or PAH-S.C. (2 ×106 cfu) 10 days
after the last immunization, and the numbers of Salmonella cells in the tumors, livers, and spleens were determined at (a) 1 and (b) 10 days postinfection. The data are
reported as means ± SD (n=4-5); n.d., not detectable. (c) Immunized mice bearing 4T1 tumors were injected i.v. with 2 ×106 cfu of S.C./Luc or PAH-S.C./Luc.
Luciferase expression levels in tissues derived from S.C./Luc-treated mice at day 1 postinfection were determined by luciferase assay. The data are reported as means ± SD (n=4) (*, p<0.05; p＜0.05 for immunized mice S.C. versus immunized mice PAH S.C.)
Figure 4. Susceptibility of mice to infection with Salmonella (S.C.). (a) Naive mice and immunized mice were injected intravenously with S.C. or PAH-S.C. (2 × 106
cfu); (a) the body weights and (b) TNF-α and (c) IL-1β levels were determined. The data are reported as means ± SD (n= 6). (*, p<0.05; **, p<0.01).
Figure 5. Antitumor effects of PAH-S.C. on immunized mice. Groups of mice were immunized i.p. with 1×104 cfu heat-killed S.C. The control mice were
immunized with PBS. Mice bearing 4T1 tumors were injected i.v. with S.C. or PAH-S.C. (2 ×106 cfu) on day 10. Vehicle control mice received PBS. (a) Tumor volumes
(mean ± SEM, n=7) in mice bearing 4T1 tumors were compared among the different treatment groups. (b) Kaplan-Meier survival curves at day 100 are shown. (p＜0.001 for naïve mice treated with vehicle versus naïve mice treated with S.C. and for naïve
immunized mice treated with S.C. versus immunized mice treated with PAH-S.C.). (**, p<0.01; ***, p<0.001). (c) Groups of mice were immunized i.p. with 1×104 cfu
PAH-S.C. The control mice were immunized with PBS. Mice bearing 4T1 tumors were injected i.v. with PAH (5 mg/ml), S.C. or PAH-S.C. (2 ×106 cfu) on day 10.
Vehicle control mice received PBS. Tumor volumes (mean ± SEM, n=7) in mice bearing 4T1 tumors were compared among different treatment groups
(**p<0.01,***p<0.001).
Figure 6. Increased numbers of infiltrating cells in tumors from immunized mice treated with PAH-S.C. Mice bearing 4T1 tumors at day 0 were injected i.v. with 2 × 106 cfu of S.C., PAH-S.C. or PBS at day 10. (a) Tumors were excised at day 20,
immunostained with antibodies against Gr-1, and Mac-3 was used to detect
infiltrating cells (200X). The numbers of (b) neutrophils and (c) macrophage cells that infiltrated the tumors were determined by averaging the cell numbers from the three fields of highest positive-stained cell density at 200X magnification in each section. The data are reported as means ± SEM (n=4); (*p<0.05); HPF, high-power field.
