Abstract

   Specific bacteria, including Fusobacterium nucleatum, Streptococcus
   sanguis, Enterococcus faecalis, and Staphylococcus xylosus, have been
   identified as contributors to breast cancer metastasis. Due to
   limitations such as lack of selectivity, traditional antibiotic
   therapies face obstacles in eliminating intratumoral bacteria. Herein,
   this work proposes the use of therapeutic vaccines to selectively
   target and eliminate harmful bacteria within tumors. A multivalent
   vaccine encapsulating both insoluble and soluble bacterial antigens was
   developed, addressing the shortcomings of traditional antibacterial
   vaccines by balancing broad antigen coverage with effective immune
   activation. This vaccine induces robust downstream immune responses to
   eliminate F. nucleatum, S. sanguis, E. faecalis, and S. xylosus,
   demonstrating notable therapeutic and preventive efficacy in
   bacteria-induced cancer metastasis models. Unexpectedly, vaccinated
   infected mice showed even slower tumor metastasis than uninfected mice.
   Overall, this study validates the potential of nanovaccines in
   modulating the intratumoral microbiome for tumor therapy and highlights
   tumor-associated bacterial infections as potential promising antitumor
   targets.
     __________________________________________________________________

   A polyvalent antibacterial vaccine could selectively kill
   tumor-associated bacteria to prevent cancer metastasis.

INTRODUCTION

   Breast cancer has emerged as one of the most prevalent malignancies
   globally, ranking among the most diagnosed cancers and a leading cause
   of cancer-related mortality in women ([40]1). While early-stage breast
   cancer, such as ductal carcinoma in situ, can be effectively managed
   through interventions such as surgical resection, the inherent loose
   cohesion between breast cancer cells and the intricate network of
   lymphatic and blood vessels within breast tissue facilitates the
   detachment and subsequent dissemination of cancer cells to distant
   organs, including the lungs, liver, and bones ([41]2). This metastatic
   journey signifies a transition to a more formidable phase of breast
   cancer, complicating treatment efforts and dimming the prospects for a
   favorable outcome ([42]3, [43]4). Consequently, beyond the pivotal role
   of early detection and intervention, thwarting the metastatic
   progression of breast cancer is of paramount importance.

   The human microbiome represents an immense ecosystem, with its
   collective genetic content surpassing that of the human genome by more
   than 150-fold ([44]5). These microbiomes, resisting across various
   bodily niches, play critical roles in both the healthy functioning and
   disease states of their host ([45]5). Recent findings have illuminated
   the presence of microorganisms within tumors, including but not limited
   to colorectal, pancreatic, and lung cancers, revealing the diverse
   bacterial profiles associated with different cancer types ([46]6). The
   microbial inhabitants of tumor tissues significantly influence cancer
   susceptibility, disease progression, and therapeutic outcomes
   ([47]7–[48]9). Specifically, in breast cancer tissues, the colonization
   by bacteria such as Fusobacterium nucleatum, Streptococcus sanguis,
   Enterococcus faecalis, and Staphylococcus xylosus has been detected
   ([49]10–[50]12). Investigation into their roles has demonstrated that
   these microorganisms can promote breast cancer metastasis through
   mechanisms such as impeding the recruitment of tumor-infiltrating T
   cells or enhancing the cytoskeletal resistance to fluid shear stress in
   tumor cells ([51]11). This burgeoning field of research underscores the
   intricate interplay between the microbiome and cancer, highlighting
   potential avenues by regulating bacteria for innovative therapeutic
   strategies.

   Antibiotics, traditionally used to eradicate bacteria, can directly
   eliminate or suppress bacteria proliferation ([52]13). Nonetheless,
   deploying antibiotics to expunge bacteria within cancer tissue is
   fraught with challenges. Humans are continuously exposed to
   environments harboring bacteria capable of colonizing tumors,
   necessitating persistent antibiotic therapy for the duration of tumor’s
   presence to prevent these bacteria from growing within the tumor
   ([53]14). This protracted use of antibiotics is particularly
   inadvisable due to several critical concerns. First, the potential side
   effects of antibiotics, including hepatorenal toxicity,
   hypersensitivity reactions, and other systemic effects, cannot be
   overlooked ([54]15). Second, antibiotics do not discriminate between
   pathogenic and symbiotic bacteria, leading to dysbiosis of the gut
   microbiota ([55]16–[56]18). This disruption can manifest in
   gastrointestinal disturbance, such as diarrhea and a compromised immune
   system, which can significantly attenuate the effectiveness of cancer
   immunotherapies such as programmed cell death protein 1 (PD-1)
   inhibitors, thus tipping the balance toward detriment rather than
   benefit ([57]19, [58]20). Moreover, the injudicious use of antibiotics
   may inadvertently fuel the rise of “superbugs” within clinical
   settings, posing a grave public health risk ([59]21, [60]22).

   To circumvent the challenges associated with antibiotic, we propose an
   innovate therapeutic strategy using vaccines to eliminate intratumoral
   bacteria, which aims to selectively target specific bacteria and
   provide long-term protection. Naturally, the uptake and presentation of
   intratumoral bacterial antigens (Ags) by immune cells are minimal,
   which often results in immune evasion by these bacteria ([61]23).
   Delivering intratumoral bacterial Ags to the immune system via vaccines
   can activate an immune response and normalize the immune surveillance
   against these bacteria ([62]24). An ideal anti-intratumoral bacterial
   vaccine should achieve the dual objectives of clearing bacteria and
   eliminating infected tumor cells ([63]25, [64]26). The key to an
   antibacterial immune response lies in activating humoral immunity to
   produce antibodies that neutralize bacteria, while the key to eliminate
   infected tumor cells lies in activating cellular immunity to enable
   cytotoxic T cells to kill these cells ([65]27, [66]28). RNA vaccines
   and peptide vaccines can induce robust cellular immunity, but their
   limited number of target proteins makes them unsuitable for bacteria
   with diverse protein ([67]29, [68]30). Inactivated vaccines with
   aluminum adjuvant, commonly used as bacterial vaccines, contain
   multiple bacterial Ags and can activate robust humoral immunity but
   have limited effects on cellular immunity ([69]31). Therefore, there is
   an urgent need for a therapeutic vaccine that can cover a broad
   spectrum of bacterial Ags while eliciting potent cellular immunity.

   To selectively target and eliminate bacteria within breast tumor cells
   and thereby suppress bacteria-induced breast cancer metastasis, we have
   developed a safe and effective quadrivalent therapeutic vaccine
   [poly(lactic-co-glycolic acid)/Ags]. This innovative vaccine
   encapsulates both water-soluble and water-insoluble components of
   sonicated lysates from F. nucleatum, S. sanguis, E. faecalis, and S.
   xylosus—the four most common metastasis-promoting bacteria found in
   human cancer tissues—into PLGA nanoparticles, which exhibit excellent
   biocompatibility ([70]32, [71]33). As a result, the vaccine boasts a
   highly abundant repertoire of bacterial Ags. The obtained PLGA/Ags are
   appropriately sized for lymph node accumulation following intradermal
   injection, where they are efficiently taken up by dendritic cells
   (DCs). Within these cells, the nanoparticles release their antigenic
   cargo, promoting DC maturation and Ag presentation, thereby eliciting
   robust humoral and cellular immunity against bacteria. In a
   bacteria-induced mouse breast cancer lung metastasis model, PLGA/Ags
   could successfully eradicate intratumoral bacteria and suppress breast
   cancer metastasis, doubling the survival time of mice with bacteria
   infected tumors. Due to the cellular immune cytotoxicity against
   infected tumors, mice treated with PLGA/Ags vaccine even exhibited
   longer survival compared to those with uninfected tumor. Furthermore,
   PLGA/Ags could induce potent immune memory effect, providing long-term
   protection against bacteria-induced breast cancer metastasis ([72]Fig.
   1). We also applied this vaccine in a bacteria-induced colon cancer
   metastasis model and verified that its efficacy can extend to other
   types of tumors. Together, the quadrivalent therapeutic vaccine
   developed here, characterized by a straightforward manufacturing
   process, robust immunogenicity, and excellent biocompatibility, holds
   promise for selectively eliminating intratumoral bacteria and
   preventing cancer metastasis in patients with bacterial infections.

Fig. 1. The schematic illustrates that the antibacterial immune response
induced by PLGA/Ags vaccine effectively prevents bacteria-induced tumor
metastasis.

   [73]Fig. 1.
   [74]Open in a new tab

   The PLGA/Ags vaccine is prepared by encapsulating with both
   water-soluble and water-insoluble bacterial lysate into a PLGA shell,
   providing a broad spectrum of Ags. Upon intradermal injection, these
   nanoparticles efficiently accumulate in the lymph nodes, facilitating
   DC maturation and antigen presentation, which subsequently triggers
   robust cellular and humoral immune responses. While uninfected tumor
   cells are prone to death in circulation, tumor cells infected by F.
   nucleatum, S. sanguis, S. xylosus, and E. faecalis can survive in the
   bloodstream and metastasize to other organs. Postvaccination, the
   specific CD8^+ cytotoxic T lymphocyte response can eradicate the
   infected tumor cells, and the humoral immunity can clear the bacteria,
   thereby preventing tumor cell metastasis. T[H] cells, T helper cells;
   T[c] cells, cytotoxic T cells. Created with [75]BioRender.com.

RESULTS

Preparation and characterization of PLGA/Ags

   To engineer a therapeutic vaccine adept at selectively eradicating
   specific bacterial populations within tumor environment, we
   meticulously designed PLGA nanoparticles encapsulating lysates from F.
   nucleatum, S. sanguis, E. faecalis, or S. xylosus. This approach was
   underpinned by the objective to emulate natural infection dynamics,
   thereby eliciting a comprehensive and enduring immune response
   ([76]34). Opting for lysates replete with a wide array of proteins from
   four bacteria served to furnish a rich antigenic palette. To extend the
   spectrum of antigenic coverage, the bacteria were lysed by
   ultrasonication in an 8 M urea solution, which was chosen for its
   superior efficiency in enhancing the solubility of insoluble proteins
   ([77]35). Subsequently, analyses including bicinchoninic acid (BCA)
   assay and SDS–polyacrylamide gel electrophoresis (PAGE) were carried
   out to confirm that bacterial lysates prepared in the 8 M urea solution
   exhibited higher protein concentrations and broader protein profile
   compared to those prepared in phosphate-buffered saline (PBS) solutions
   ([78]Fig. 2, A and B).

Fig. 2. Preparation and characterization of PLGA/Ags nanovaccines.

   [79]Fig. 2.
   [80]Open in a new tab

   (A) Protein concentration of lysates obtained from F. nucleatum, S.
   sanguis, E. faecalis, and S. xylosus after sonication in urea and PBS
   solutions. US, Ultrasonic lysis. (B) SDS–polyacrylamide gel
   electrophoresis (PAGE) analysis of proteins from markers, and
   ultrasonic lysates of F. nucleatum, S. sanguis, E. faecalis and S.
   xylosus in urea and PBS solutions. (C) Hydrodynamic sizes of PLGA/Ags
   nanoparticles before and after freeze-dry. (D and E) Transmission
   electron microscopy (TEM) images of PLGA/Ags nanoparticles before and
   after freeze-drying (scale bars, 0.5 μm). (F) Time-lapse hydrodynamic
   size and PDI of PLGA/Ags after freeze-drying. Data are presented as the
   means ± SD (n = 3). (G) SDS-PAGE analysis of proteins before and after
   encapsulation in PLGA. (H) Number of protein species detected by liquid
   chromatography–mass spectrometry (LC-MS) in F. nucleatum, S. sanguis,
   E. faecalis, and S. xylosus before and after encapsulation in PLGA. (I)
   Proportions of primary subcellular locations of proteins from PLGA/Ags
   nanoparticles. (J) Word cloud illustrating subcellular locations of
   proteins from PLGA/Ags nanoparticles.

   Then, the obtained bacterial lysates were encapsulated into PLGA
   nanoparticles (PLGA/Ags) using a double-emulsion technique followed by
   solvent evaporation. Different feed ratios of PLGA and bacterial
   lysates were tested (table S1), with the final formulation chosen on
   the basis of a bacterial Ag loading capacity of 3.33%. The resultant
   nanoparticles were measured by dynamic light scattering (DLS), which
   revealed a hydrodynamic diameter of ~200 nm ([81]Fig. 2C). To ensure
   effective Ag delivery to Ag-presenting cells (APCs), it is essential
   that vaccine nanoparticles maintain stability under in vivo conditions
   and prevent the premature release of Ags into body fluids. To
   investigate the stability of PLGA/Ags, it was incubated in PBS at 37°C
   for 21 days. No significant changes were observed in the average
   hydrodynamic size and the polydispersity index (PDI) (fig. S1),
   indicating that the formulation remains stable under conditions that
   closely resemble the in vivo environment. Subsequently, transmission
   electron microscopy (TEM) provided detailed imaging to confirm the
   homogenous size distribution and spherical morphology of PLGA/Ags
   ([82]Fig. 2D). Recognizing the logistical challenges posed by the
   necessity for cold chain management in current vaccine dissemination,
   which can impede global accessibility, there is a pressing need to
   forge vaccines amenable to room temperature storage ([83]36, [84]37).
   In our endeavor to surmount this hurdle, sucrose was used as a
   cryoprotectant during the freeze-drying process of PLGA/Ags ([85]38).
   As shown in [86]Fig. 2 (C and E), DLS and TEM analyses indicated that
   the lyophilization and reconstitution did not affect the size and
   morphology of the PLGA/Ags nanoparticles. Moreover, the average
   hydrodynamic size and PDI of the PLGA/Ags remained consistency after
   freeze-drying and subsequent reconstitution, even after intervals of 90
   and 180 days ([87]Fig. 2F), demonstrating the exceptional stability of
   PLGA/Ags at ambient conditions, heralding implications for storage and
   distribution without reliance on cold chain logistics.

   To evaluate whether various bacterial proteins are all encapsulated in
   PLGA, SDS-PAGE was performed. As shown in [88]Fig. 2G, a direct
   correlation was observed between the protein bands from the bacterial
   lysate and those evident in the PLGA encapsulated group, indicating the
   successful entrapment of a diverse array of proteins derived from the
   bacterial lysates within the PLGA nanoparticles. Furthermore, a
   comprehensive proteomic analysis using liquid chromatography with
   tandem mass spectrometry (LC-MS/MS) identified a total 3642 proteins in
   the PLGA/Ags complexes. These results confirmed that the protein
   profile of PLGA encapsulates mirrors that of the original bacterial
   lysates, encompassing an extensive spectrum of proteins from each
   bacterium species ([89]Fig. 2H). Further insights gained from the
   subcellular localization analysis of the proteins identified by
   LC-MS/MS indicated that these predominantly hail from the cytoplasm,
   plasma membrane, membrane, and ribosomes, suggesting that the antigenic
   repertoire within the PLGA nanoparticles comprehensively represents
   bacterial proteins across various cellular domains ([90]Fig. 2, I and
   J). Because F. nucleatum, S. sanguis, E. faecalis, and S. xylosus are
   intracellular bacteria, proteins at various locations within these
   bacteria can be processed by tumor cells and presented on the cell
   surface via major histocompatibility complex I (MHC I). Using PLGA/Ags
   to activate immune responses that cover a broad range of Ags can
   generate diverse CD8^+ T cells targeting these Ags, thereby reducing
   immune evasion by tumor cells harboring these bacteria.

In vitro immune stimulation by PLGA/Ags

   For a therapeutic vaccine, the capacity to activate DC maturation is
   crucial for Ag presentation and subsequent immune system engagement
   ([91]39). Therefore, in vitro assessments of the influence exerted by
   PLGA/Ags complexes on DC maturation were conducted ([92]Fig. 3A).
   Before the principal investigations, we ascertained the biosafety
   profile of our PLGA/Ags formulation. At the protein concentration up to
   250 μg/ml, the DC2.4 cells still maintained good viability in the
   presence of PLGA/Ags (fig. S2), indicating the successful mitigation of
   potential cytotoxic components, such as lipopolysaccharides (LPSs) in
   the bacterial lysates, during the encapsulation process. For the
   maturation of DCs in vitro, bone marrow–derived DCs (BMDCs) exposed to
   PLGA/Ags demonstrated the most marked elevation in the expression of
   co-stimulatory molecules (CD80 and CD86) compared to both the untreated
   and free Ag–treated cells ([93]Fig. 3, B and C). Additionally, BMDCs in
   the company of PLGA/Ags secreted significantly higher levels of
   pro-inflammatory cytokines, including interleukin-12p70 (IL-12p70),
   tumor necrosis factor–α (TNF-α), and IL-6 ([94]Fig. 3, D to F).
   Additionally, PLGA/Ags nanoparticles subjected to long-term
   lyophilization were shown to retain their ability to effectively
   promote DC maturation (fig. S3, A and B). This superior performance of
   PLGA/Ags in stimulating DC maturation may be attributed to the
   augmented internalization of nanoparticles ([95]40). We then
   investigated the internalization of free Ags and PLGA/Ags by DC2.4
   cells. As illustrated in [96]Fig. 3 (G and H), the Ag internalization
   by DCs was substantially higher in the PLGA/Ags group than that in the
   free Ags group, a gap that widened with time. Considering that the
   activation of the immune response hinges not only on the Ag acquisition
   but also on the processing and presentation of these Ags ([97]41), we
   used confocal microscopy to track the journey of fluorescein
   isothiocyanate (FITC)–labeled Ags within DCs. Between 4 and 8 hours,
   there was a progressive intensification of the Ag signal, which
   coalesced with the lysosome signal. From 12 to 24 hours, we observed
   Ags breaking free from lysosomal confines and arranging themselves on
   the cell surface in a ring-like pattern ([98]Fig. 3I and fig. S4). At
   each observed time point, the Ag signals in the PLGA/Ags group
   overshadowed that in the free Ags group, indicating that Ags loading
   onto PLGA nanoparticles effectively increased Ag uptake and
   presentation by DCs.

Fig. 3. In vitro DC uptake and activation.

   [99]Fig. 3.
   [100]Open in a new tab

   (A) Schematic representation of the experimental setup for DC uptake
   and activation. Created with [101]BioRender.com. (B and C)
   Representative flow cytometric plots (B) and statistical analysis (C)
   showing the maturation of BMDCs in different groups. Data are presented
   as means ± SD (n = 3). (D to F) Secretion levels of interleukin-12p70
   (IL-12p70) (D), tumor necrosis factor–α (TNF-α) (E), and IL-6 (F) by
   BMDCs in different groups. Data are presented as means ± SD (n = 3). (G
   and H) Representative flow cytometric plots (G) and statistical
   analysis (H) of mean fluorescence intensity (MFI) in DC2.4 cells
   incubated with fluorescein isothiocyanate (FITC)–labeled free Ags or
   PLGA/Ags for different time points. Data are presented as means ± SD (n
   = 3). (I) Confocal fluorescence images of DC2.4 cells incubated with
   FITC-labeled (green) free Ags or PLGA/Ags for different time points.
   Nuclei and lysosomes were stained with 4′,6-diamidino-2-phenylindole
   (DAPI; blue) and LysoTracker (red), respectively (scale bars, 40 μm).
   h, hours. (J) Heatmap showing the transcript expression of the related
   genes in BMDCs incubated with PLGA/Ags or PBS. (K) Kyoto Encyclopedia
   of Genes and Genomes pathway analysis based on differentially expressed
   genes between the PLGA/Ags and untreated groups. NF-κB, nuclear factor
   κB; ECM, extracellular matrix; NOD, nucleotide oligomerization domain;
   JAK-STAT, Janus kinase–signal transducer and activator of
   transcription. (L) Gene ontology analysis based on differentially
   expressed genes. Statistical significance was calculated via one-way
   analysis of variance (ANOVA) [(C) to (F)] and unpaired t test (H) in
   GraphPad Prism. NK, natural killer; *P < 0.05; **P < 0.01; ***P <
   0.001; ****P < 0.0001.

   To delve deeper into the mechanism of immune activation induced by
   PLGA/Ags, we conducted transcriptome sequencing on BMDCs cultured
   alongside either PLGA/Ags or PBS for comparative analysis. Genes
   implicated in BMDC maturation, including those coding for TNFs, ILs,
   chemokines, and their receptors, were significantly up-regulated after
   PLGA/Ags treatment ([102]Fig. 3J). In addition, through Kyoto
   Encyclopedia of Genes and Genomes pathway enrichment analysis, it was
   found that the up-regulated differentially expressed genes were
   predominantly concentrated in pathways such as Toll-like receptor (TLR)
   signaling, nuclear factor κB signaling, C-type lectin receptor
   signaling, and nucleotide oligomerization domain–like receptor
   signaling ([103]Fig. 3K), revealing that the activation of BMDCs
   induced by PLGA/Ags is a composite process involving multiple signaling
   cascades. Furthermore, gene ontology analysis revealed that the
   up-regulated differential expression genes were highly associated with
   processes including T cell activation, T helper 1–type immune response
   and the secretion of inflammatory cytokines ([104]Fig. 3L), suggesting
   that the DCs activated by PLGA/Ags have the enhanced capability to
   efficiently activate downstream immune response.

In vivo immune stimulation by PLGA/Ags

   Different from the in vitro experiments where nanoparticles can
   directly interact with DCs in substantial quantities, Ags administrated
   intradermally must navigate to the draining lymph nodes (dLNs) to
   engage with DCs ([105]42). Before investigating the immune response
   elicited by the PLGA/Ags vaccine, its transportation efficacy to the
   dLNs was scrutinized. As shown in [106]Fig. 4 (A to C), sequential
   imaging of BALB/c mice, intradermally injected with either
   PLGA/Ag-Cy5.5 or free Ag–Cy5.5, revealed distinct patterns over time.
   The fluorescence intensity within the inguinal dLNs of mice injected
   with PLGA/Ag-Cy5.5 markedly increased, peaking at 8 hours and
   subsequently diminishing, yet remained discernible up to 36 hours
   postinjection. Conversely, the fluorescence intensity in the inguinal
   dLNs of mice receiving free Ag–Cy5.5 consistently remained low,
   potentially attributable to the lymphatic system’s tendency to
   preferentially transport nanoparticles in the 100- to 200-nm-size
   range, while smaller, free proteins may bypass this mechanism
   ([107]43).

Fig. 4. In vivo immune stimulation by PLGA/Ag nanovaccine.

   [108]Fig. 4.
   [109]Open in a new tab

   (A) Schematic and timeline depicting the experimental design to
   evaluate the ability of PLGA/Ags to accumulate in lymph nodes. i.d.,
   intradermal. Created with [110]BioRender.com. (B) In vivo time-lapse
   fluorescence images of dLNs in mice treated with free Ag–Cy5.5 or
   PLGA/Ag-Cy5.5. (C) Statistical analysis of average Cy5.5 fluorescence
   signals in dLNs of mice based on images in (B). (D) Confocal
   fluorescence images of dLNs in mice treated with free Ag–Cy5.5 or
   PLGA/Ag-Cy5.5 (scale bars, 250 μm). (E) MFI of Cy5.5 in DC subsets from
   dLNs in mice treated with free Ag–Cy5.5 or PLGA/Ag-Cy5.5. cDCs,
   conventional DCs; pDCs, plasmacytoid DCs. (F) Schematic and timeline
   illustrating the experimental design to evaluate immune responses
   triggered by PLGA/Ags vaccine. (G to I) Expression levels of (G) CD80,
   CD86, (H) CD40, and (I) MHC II on DCs, indicating the maturation of DCs
   in the dLNs of mice injected with different vaccines. (J and K)
   Percentages of (J) CD3^+ and (K) CD3^+CD8^+ T cells in the dLNs of mice
   injected with different vaccines. (L) Percentages of IFN-γ^+CD8^+ T
   cells within CD8^+ T cells collected from mice in different groups
   after restimulated with bacterial Ags. (M) Percentages of IFN-γ^+CD8^+
   T cells within CD8^+ T cells collected from mice treated with PLGA/Ags
   vaccines after restimulated with different bacterial Ags. (N and O)
   Percentages of (N) CD3^+CD4^+ T cells and (O) CD19^+B220^+ B cells in
   the dLNs of mice in different groups. (P) Anti-bacteria IgG titers in
   the serum of mice injected with different vaccines. All data are
   presented as means ± SD (n = 4). Statistical significance was
   calculated via unpaired t test (M) and one-way ANOVA [(G) to (P)] in
   GraphPad Prism. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

   Following the validation of the effective intralymphatic delivery of
   PLGA/Ags, we delved into the uptake of PLGA/Ags by APC) within dLNs.
   Confocal imaging of dLNs sections revealed that the Cy5.5 fluorescence
   signal was higher in the PLGA/Ag-Cy5.5 group compared to that in the
   free Ag–Cy5.5–treated group. Notably, regions exhibiting Cy5.5
   fluorescence signal were devoid of 4′,6-diamidino-2-phenylindole (DAPI)
   fluorescence in the free Ag–Cy5.5–treated group, whereas, in the
   PLGA/Ag-Cy5.5–treated group, Cy5.5 fluorescence signals co-localized
   with DAPI signal, indicating that PLGA/Ags not only accumulate in dLNs
   but also proficiently convey Ags to APCs ([111]Fig. 4D). To further
   dissect the dynamics of Ag uptake within dLNs, the specific DC subsets
   engaging in this process were further characterized using flow
   cytometry. As shown in [112]Fig. 4E, it was observed that across DC
   subsets, more cells internalized the Ags in the PLGA/Ag-Cy5.5 group
   relative to the free Ag–Cy5.5–treated group. Intriguingly, in the
   PLGA/Ag-Cy5.5 group, CD8^+ conventional DCs, which play a crucial role
   in T cell activation and Ag internalization ([113]44), followed by
   plasmacytoid DCs, which can rapidly secrete large amounts of interferon
   (IFN) in response to intracellular pathogen infections ([114]45),
   internalized more Ags. In contrast, tolerogenic CD11b^−CD103^+ DCs
   internalized less Ags ([115]46). This comprehensive evaluation
   underscored the proficiency of PLGA vaccine in not only reaching the
   dLNs but also in facilitating efficient Ag uptake by key immune
   players, paving the way for potent immune activation.

   Subsequently, the maturation status of DCs in vivo prompted by the
   vaccine, along with other immune parameters, was validated using a
   murine model ([116]Fig. 4F). The dLNs, spleens, and peripheral blood
   harvested from mice with two doses of vaccine (10 μg of Ag per mouse
   per dose) were processed into single-cell suspensions for comprehensive
   flow cytometry analysis ([117]47). As anticipated, in comparison with
   the untreated group, the free Ags group, and the group receiving a
   traditional aluminum adjuvant combined with an inactivated bacterial
   vaccine (Al + IV), mice vaccinated with PLGA/Ags exhibited elevated
   expression levels of co-stimulatory molecule (CD80, CD86, CD40, and MHC
   II) on DCs within their dLNs ([118]Fig. 4, G to I, and fig. S5A).
   Following this, the proportion of T cells in the dLNs, which are
   recipients of Ag presentation by DCs, was assessed ([119]41). The
   results in [120]Fig. 4J and fig. S5B indicated that T cells (CD3^+)
   were more abundant in mice vaccinated with PLGA/Ags compared to those
   in other groups. Given the intracellular nature of F. nucleatum, S.
   sanguis, E. faecalis, and S. xylosus, a strong cellular immune response
   is imperative to eradicate infected tumor cells and eliminate these
   bacteria ([121]11, [122]23, [123]48). As shown in [124]Fig. 4K and fig.
   S5C, the proportion of CD8^+ T cells was significantly higher in mice
   vaccinated with PLGA/Ags compared to that in other groups. To further
   confirm the Ag-specific cellular immune response of CD8^+ T cells,
   splenocytes from vaccinated mice were rechallenged with bacterial cell
   membranes in vitro. As expected, CD8^+ T cells from mice treated with
   PLGA/Ags secreted significantly higher levels of IFN-γ when stimulated
   with Ags from the four bacteria simultaneously, compared to other
   groups ([125]Fig. 4L and fig. S5D). Meanwhile, CD8^+ T cells from
   PLGA/Ags-treated mice also produced substantial amounts of IFN-γ when
   stimulated with each bacterial Ag separately ([126]Fig. 4M).

   In addition to cellular immune response, humoral immunity also plays an
   important role in eliminating bacteria by neutralizing extracellular
   bacteria and those released from infected cells upon destruction with
   antibodies ([127]34). As shown in [128]Fig. 4 (N and O) and fig. S5 (E
   and F), the proportions of CD4^+ T cells and B cells (CD19^+B220^+)
   were significantly increased in mice vaccinated with PLGA/Ags. To
   directly quantify humoral immune activation, the antibody titers
   against bacterial Ags in the peripheral blood serum were quantified
   using enzyme-linked immunosorbent assay (ELISA) on Ag-coated plates.
   Mice vaccinated with PLGA/Ags exhibited significantly superior antibody
   production compared to the other groups ([129]Fig. 4P). Thus, although
   the traditional aluminum adjuvant combined with inactivated vaccines
   enhanced DC activation when compared to free Ags, it showed limited
   efficiency in activating cellular immunity and humoral immunity.
   Conversely, the PLGA/Ags vaccine induced significantly stronger DC
   activation, as well as both cellular and humoral immune responses
   compared to the other groups. Given the strong stimulatory effect of
   PLGA/Ags, we further conducted a thorough biosafety evaluation. On day
   6, after two rounds of vaccination, blood samples were collected for
   comprehensive hematological analysis, and major organ tissues were
   subjected to hematoxylin and eosin (H&E) staining for histopathological
   assessments. As shown in fig. S6A, all blood biochemistry and
   hematological parameters from mice vaccinated with PLGA/Ags remained
   within the normal reference range. Additionally, as shown in fig. S6B,
   no significant pathological changes were observed in the major organs
   of mice treated with PLGA/Ags. These results demonstrate that PLGA/Ags
   have negligible side effects on normal tissues and organs, supporting
   its excellent safety profile as a vaccine.

PLGA/Ags prevent cancer metastasis by eliminating intratumoral bacteria

   To ascertain the therapeutic potential of PLGA/Ags in eradicating
   intratumoral bacteria and thwarting bacteria-induced tumor metastasis,
   investigative studies were expanded to encompass tumor-bearing mice
   models. The BALB/c mice were allocated into five distinct groups: (i)
   intravenous injection of luciferase-transfected 4T1 (4T1-Luc) tumor
   cells unafflicted by bacterial infection (uninfected); (ii) intravenous
   injection of tumor cells infected with a consortium of F. nucleatum, E.
   faecalis, S. sanguis, and S. xylosus (bacteria); (iii) intravenous
   injection of tumor cells with bacterial infection followed by treatment
   with free bacterial Ags (free Ags); (iv) intravenous injection of tumor
   cells with bacterial infection followed by vaccination with aluminum
   adjuvant and inactivated bacterial vaccine (Al + IV); and (v)
   intravenous injection of tumor cells with bacterial infection followed
   by vaccination with PLGA/Ags. Each mouse was injected with 2.5 × 10^5
   4T1-Luc cells, with infected tumor cells at a multiplicity of infection
   (MOI) of 100, and the vaccine was administered at a dose of 10 μg of Ag
   per mouse. Once 4T1-Luc pulmonary metastases reach a certain size, they
   can emit bioluminescence upon exposure to luciferase substrates. In
   vivo imaging was performed at 6-day intervals to monitor the
   progression of pulmonary metastases ([130]Fig. 5A). Consistent with the
   prior results, the data depicted in [131]Fig. 5 (B and C) illustrated
   that mice in the bacteria group experienced abbreviated survival
   periods and accelerated metastatic dissemination compared to the
   uninfected group, confirming the potent tumor metastasis induced by F.
   nucleatum, E. faecalis, S. sanguis, and S. xylosus. Among the treatment
   groups, administration of free Ags was insufficient to prevent tumor
   metastasis. Similarly, the Al + IV treatment also did not appreciably
   curtail metastatic progression to levels akin to those observed in the
   absence of bacteria. In stark contrast, mice that received the PLGA/Ags
   vaccination exhibited significantly extended survival periods compared
   to the bacteria group, highlighting the pronounced efficacy of the
   PLGA/Ags vaccine in enhancing host resistance against bacteria-driven
   tumor progression and metastasis. Unexpectedly, the survival period of
   the PLGA/Ags-treated group even surpassed that of the uninfected group,
   which may be attributed to the fact that tumor cells infected with
   intracellular bacteria present bacterial peptides on their surface via
   MHC I molecules, making them susceptible to cytotoxic T cells that
   recognize bacterial epitopes generated by the vaccine, in addition to
   those that recognize tumor epitopes ([132]23, [133]24). Moreover,
   previous studies have demonstrated the presence of shared antigenic
   epitopes between host cells and bacteria, which could also contribute
   to the antitumor effects of the antibacterial vaccine ([134]49).
   Therefore, PLGA/Ags not only eliminates specific bacteria to inhibit
   bacteria-driven tumor progression and metastasis but also enhances
   antitumor immune responses, thereby delaying the growth of cancer
   cells.

Fig. 5. PLGA/Ags overcoming bacteria-induced breast tumor metastasis.

   [135]Fig. 5.
   [136]Open in a new tab

   (A) Schematic and timeline illustrating the experimental design to
   evaluate the ability of PLGA/Ags in preventing bacteria-induced tumor
   metastasis. (B) Survival curves of mice in different groups (n = 6).
   (C) In vivo bioluminescence imaging to track the metastasis of 4T1-Luc
   cancer cells in mice with different treatments (n = 6). (D) Schematic
   and timeline showing the experimental design to evaluate the selective
   antibacterial activity of PLGA/Ags. i.v., intravenous. (E)
   Representative colony plate images showing the bacteria abundance in
   homogenized lungs of mice with different treatments. (F to I)
   Representative (F) hematoxylin and eosin (H&E) staining, (G) Ki-67
   staining (scale bar, 4 mm), (H) gross morphology (scale bar, 1 cm), and
   (I) bioluminescence images of lungs in indicating different groups. (J)
   Colony-forming units (CFUs) quantifying bacteria abundance in
   homogenized lungs of mice after different treatments. Data are
   presented as means ± SD (n = 3). (K) Average bioluminescence signals of
   lungs in mice with different treatments. Data are presented as means ±
   SD (n = 3). (L) Relative abundance of lung microbiome in mice with or
   without PLGA/Ags treatment. Family-level taxonomy is presented as the
   percentage of total sequences (n = 3). (M) CFUs showing the bacteria
   abundance in homogenized lungs of mice with different treatments. Data
   are presented as the means ± SD (n = 6). n.s., not significant. (N)
   Schematic representation of cellular immunity and humoral immunity in
   clearing infected tumor cells and bacteria. Statistical significance
   was calculated via the log-rank (Mantel-Cox) test (B) and unpaired t
   test (C) in GraphPad Prism. *P < 0.05; ***P < 0.001. (A), (D), and (N)
   created with [137]BioRender.com.

   To comprehensively demonstrate the antibacterial effect of PLGA/Ags to
   delay the growth of tumor, a new round of treatment was conducted with
   the same experimental groups as described above ([138]Fig. 5D). On the
   12th day, all mice were euthanized, and their lungs were harvested for
   a series of analyses. As shown in [139]Fig. 5 (E and J), the lung
   homogenate plating results indicated a reduction in lung bacterial
   colonies in the PLGA/Ags-treated mice, approaching levels observed in
   the uninfected group. As illustrated in [140]Fig. 5 (F to H), H&E
   staining and Ki-67 staining of lung sections from the mice demonstrated
   substantially smaller dark tumor-indicative areas in the PLGA/Ags group
   compared to those in the bacteria group. This group also outperformed
   the free Ags group and the Al + IV group, nearly reaching the levels of
   the uninfected group. Lung images ([141]Fig. 5H and fig. S7A) and
   bioluminescence imaging ([142]Fig. 5, I and K, and fig. S7B) results
   corroborated these findings. To accurately reflect that the bacteria
   cleared by the vaccine were the tumor-associated F. nucleatum, E.
   faecalis, S. sanguis, and S. xylosus, bacterial community sequencing
   was performed on the lungs of the PLGA/Ags and bacteria groups. As
   shown in [143]Fig. 5L and fig. S8, PLGA/Ags treatment effectively
   reduced the abundance of Fusobacterium, Enterococcus, Streptococcus,
   and Staphylococcus in the lungs of the mice. To investigate the role of
   humoral and cellular immunity, anti-CD4 or anti-CD8 antibodies were
   administered to mice during vaccination to block either humoral or
   cellular immunity. As shown in [144]Fig. 5M and fig. S9, the bacterial
   count in the PLGA/Ags group was significantly lower compared to that in
   the CD4 block and CD8 block groups, indicating that both cellular and
   humoral immunity are essential components of the antibacterial immune
   response within tumors ([145]Fig. 5N). Together, these experimental
   results indicate that the specific immunity induced by PLGA/Ags
   effectively eliminated F. nucleatum, E. faecalis, S. sanguis, and S.
   xylosus within tumor cells, thereby preventing the tumor lung
   metastasis induced by these bacteria.

   Building on the above results, we further applied PLGA/Ags to a
   bacteria-induced CT-26 tumor metastasis model. On day −1, mice were
   intravenously injected with 2.5 × 10^5 CT-26-Luc cells, either infected
   or uninfected with bacteria, to establish the model. On days 0 and 6,
   the PLGA/Ags group was intradermally injected with a vaccine containing
   10 μg of Ag. As shown in fig. S10 (A and B), F. nucleatum, E. faecalis,
   S. sanguis, and S. xylosus were also found to promote the metastasis of
   CT-26-Luc cancer cells. Tumor progression in bacteria-infected mice
   vaccinated with PLGA/Ags was significantly delayed compared to that in
   bacteria-infected mice and was also slightly delayed relative to
   uninfected mice. This result was consistent with observations from the
   4T1-Luc model, further demonstrating that the immune response triggered
   by the antibacterial vaccine can effectively eliminate
   bacteria-infected tumor cells and delay cancer metastasis.

The long-term protective effect induced by PLGA/Ags

   Breast cancer has a high propensity for both metastasis and recurrence,
   with recurrent tumors being particularly prone to further metastasis
   ([146]50). Recent studies demonstrated that specific bacteria can
   induce tumor metastasis during the initial stage and also promote
   metastasis upon recurrence ([147]51). Therefore, vaccines targeting
   intratumoral harmful bacteria must not only elicit a short-term immune
   response upon injection but also establish long-term immune memory
   effect to prevent bacteria from recolonizing recurrent tumor tissues
   and promoting tumor progression. Consequently, whether PLGA/Ags can
   induce long-term antibacterial immunity has emerged as a new area of
   interest. BALB/c mice were divided into two groups: (i) untreated, and
   (ii) PLGA/Ags (intradermally on days 0, 7, and 14). Each mouse received
   a vaccine dose containing 10 μg of Ag. Then, the blood samples were
   collected on days 7, 14, 28, and 42 ([148]Fig. 6A). As shown in
   [149]Fig. 6B, the Ag-specific immunoglobulin G (IgG) levels in blood
   serum, assessed by ELISA using plates coated with Ags from F.
   nucleatum, S. sanguis, E. faecalis, or S. xylosus, demonstrated that
   PLGA/Ags induced humoral immunity against all target bacteria. The
   total anti-bacteria IgG titers in the PLGA/Ags group were tracked over
   time, showing a gradual increase from days 7 to 28, followed by a
   decline ([150]Fig. 6C). On day 42, peripheral blood was sampled to
   detect memory immune cells. As shown in [151]Fig. 6 (D to F),
   significant increases were observed in both effector memory T cells
   (CD44^+CD62L^−) and central memory T cells (CD44^+CD62L^+) in mice
   vaccinated with PLGA/Ags. Additionally, the proportion of memory B
   cells (CD19^+CD38^−) in the vaccinated mice exhibited a significant
   increase ([152]Fig. 6G).

Fig. 6. Long-term immune memory induced by PLGA/Ags.

   [153]Fig. 6.
   [154]Open in a new tab

   (A) Schematic and timeline illustrating the experimental design to
   evaluate the ability of PLGA/Ags to induce immune memory effect in
   preventing bacteria-induced tumor metastasis. Created with
   [155]BioRender.com. (B) IgG titers in the serum of mice with or without
   PLGA/Ags vaccine treatment against different bacteria. Data are
   presented as means ± SD (n = 3). (C) Anti-bacteria IgG titers in the
   serums of mice injected with PLGA/Ags vaccines. Data are presented as
   means ± SEM (n = 3). (D to F) Representative flow cytometric plots (D)
   and statistical analysis of (E) effector memory T cells (T[EM])
   (CD44^+CD62L^−) and (F) central memory T cells (T[CM]) (CD44^+CD62L^+)
   within CD8^+ T cells of mice with or without PLGA/Ags vaccine
   treatment. Data are presented as means ± SD (n = 3). (G) CD19^+CD38^−
   memory B cells within CD45^+ lymphocytes of mice with or without
   PLGA/Ags vaccine treatment. Data are presented as means ± SD (n = 3).
   (H) In vivo bioluminescence imaging to track metastasis of 4T1-Luc
   cancer cells in mice with different treatments (n = 3). (I to K)
   Representative (I) bioluminescence images, (J) H&E staining slides, and
   (K) Ki-67 staining slides (scale bar, 5 mm) of lungs in different
   groups. (L) Average bioluminescence signals in the lungs of mice with
   different treatments. Data are presented as means ± SD (n = 3 for
   uninfected group and PLGA/Ags group and n = 2 for the bacteria group
   due to mouse mortality). Statistical significance was calculated via
   unpaired t test (E) and (G) in GraphPad Prism. *P < 0.05.

   Following three doses of PLGA/Ags vaccine, the mice were intravenous
   injected with 4T1-Luc cells infected with F. nucleatum, S. sanguis, E.
   faecalis, and S. xylosus (2.5 × 10^5 4T1-Luc cells per mouse, with an
   MOI of 100). Meanwhile, unvaccinated mice injected with infected or
   uninfected tumor cells were used as the control. The lung metastases
   were diligently tracked via the bioluminescence emitted by the
   luciferase with 6-day intervals ([156]Fig. 6H). It was observed that
   three doses of PLGA/Ags effectively delayed the rate of pulmonary
   metastasis in bacteria-infected tumors, which is even a slower
   progression compared to that in tumors not infected with bacteria.
   Then, the mice were euthanized on day 55, the bioluminescence imaging
   ([157]Fig. 6, I and L) and histological analysis of lungs ([158]Fig. 6,
   J and K) further illustrated the antibacterial immune response induced
   by PLGA/Ags, preventing the progression of metastasis. In summary, in
   long-term immunity experiments, PLGA/Ags demonstrated its ability to
   establish effective immune memory, providing prolonged antibacterial
   protection and preventing bacteria-induced tumor metastasis. Moreover,
   this immune memory enhances the body’s ability to eliminate
   bacteria-infected tumor cells more effectively than uninfected tumor
   cells.

DISCUSSION

   In conclusion, this study developed a multivalent therapeutic
   antibacterial vaccine (PLGA/Ags) by encapsulating both soluble and
   insoluble fractions of lysates from F. nucleatum, S. sanguis, E.
   faecalis, and S. xylosus within PLGA nanoparticles. This vaccine aims
   to selectively eliminate these bacteria and tumor cells infected by
   them, thereby preventing bacteria-induced breast cancer metastasis. It
   helps prevent damage to normal microbiota and the development of
   antibiotic-resistant bacteria, which can result from the prolonged use
   of antibiotics to eliminate intratumoral bacteria. PLGA/Ags carry a
   rich array of Ags, enabling the immune system to comprehensively
   recognize the four target bacteria, and elicit a robust cellular immune
   response, allowing cytotoxic T cells to kill tumor cells infected with
   the target bacteria. The vaccine demonstrated significant efficacy in
   both the treatment and prevention of breast cancer lung metastasis
   models. It not only counteracted the tumor-promoting effects of the
   bacteria but also slowed tumor progression in vaccinated,
   bacteria-infected mice compared to that in uninfected mice. This study
   demonstrates that modulating the tumor microbiome with an antibacterial
   vaccine can combat breast cancer metastasis, highlighting the critical
   role of the tumor microbiome in cancer therapy. As more
   tumor-associated microbial species are identified, this vaccine
   platform can be adapted to selectively eliminate bacteria detrimental
   to cancer treatment while sparing beneficial ones, providing diverse
   solutions for various cancer types. Additionally, this study suggests
   that intratumoral bacteria could serve as targets for antitumor
   strategies, offering a perspective for developing innovative cancer
   therapies with potential clinical value.

MATERIALS AND METHODS

Materials, bacterial strains, cells, and animals

   PLGA with Mw of 12,000 to 15,000 (ester terminated, 50:50) was
   purchased from J&K Scientific. Poly(vinyl alcohol) (PVA; 80%
   hydrolyzed, Mw of 9000 to 10,000) was obtained from Sigma-Aldrich. F.
   nucleatum [American Type Culture Collection (ATCC), 25586] was acquired
   from the China General Microbiological Culture Collection Center and
   cultured in thiolglycollate broth in an anaerobic environment. E.
   faecalis (ATCC, 29212) was procured from the Guangdong Microbial
   Culture Collection Center and cultured in brain heart infusion broth
   medium. S. sanguis (ATCC, 10556) was also obtained from the Guangdong
   Microbial Culture Collection Center and cultured in thiolglycollate
   medium. S. xylosus (ATCC, 29971) was sourced from the same center and
   cultured in Luria-Bertani medium. All bacterial solid culture media
   were prepared using Columbia blood agar. All media for bacteria culture
   were purchased from Qingdao Hope Bio-Technology Co. Ltd.

   4T1-Luc cells were obtained from Shanghai Genechem Co. Ltd. and were
   cultured in RPMI 1640 medium (6016011, Dakewe Biotech) containing 10%
   fetal bovine serum (FBS) (JYK-FBS-301, Inner Mongolia Jin Yuan Kang
   Biotechnology Co. Ltd.) and 1% penicillin/streptomycin ([159]PB180120,
   Pricella Life Science &Technology Co. Ltd.) at 37°C in 5% CO[2]. BMDCs
   were prepared from C57BL/6 mice, and cultured in RPMI 1640 medium
   supplemented with 10% FBS, 1% penicillin/streptomycin, 0.1%
   β-mercaptoethanol, 0.01% granulocyte-macrophage colony-stimulating
   factor (CK-02, Novoprotein, Shanghai, China) and 0.01% IL-4
   ([160]HA210902, HUABIO). DC2.4 cells was purchased from Pricella Life
   Science & Technology Co. Ltd.

   Six- to 8-week female BALB/c mice and 6- to 8-week C57BL/6 mice were
   purchased from Changzhou Cavens Laboratory Animal Co. Ltd. All animal
   experiments were performed in compliance with the relevant laws and
   approved by the Institutional Animal Care and Use Committee of Soochow
   University (no. SUDA20240724A01).

Bacterial Lysate Preparation

   F. nucleatum, E. faecalis, S. sanguis, and S. xylosus were cultured at
   37°C for 48 hours. Subsequently, bacterial cells were harvested by
   centrifugation at 10,000g for 10 min, followed by three sequential
   washes with PBS. The bacteria from each 50 ml of culture medium were
   then resuspended in 1 ml of 8 M urea solution and underwent sonication
   using a Sonic Dismembrator for 30 min on ice (sonication cycle: T[on] =
   3 s and T[off] = 5 s; ultrasonic power of 500 W). Following
   ultrasonication, the suspensions were centrifuged at 10,000g and 4°C
   for 10 min, and the resulting supernatants were collected. The protein
   concentration of bacterial lysates was determined using BCA protein
   assay kit (AKPR017, Beijing Boxbio Science & Technology Co. Ltd.). The
   protein compositions were then analyzed by SDS-PAGE (EC0022-A, Shandong
   Sparkjade Biotechnology Co.) and stained by Coomassie brilliant blue.

Synthesis and characterization of PLGA/Ags

   The PLGA nanoparticles were synthesized by the double-emulsion method.
   Briefly, 0.3 ml of bacterial lysate supernatant (protein at 1 mg/ml for
   each bacterial type) was added to 1 ml of dichloromethane solution
   containing PLGA (5 mg/ml). This mixture was homogenized by ultrasound
   to generate a water-in-oil (W/O) emulsion. Then, 1.3 ml of the W/O
   emulsion was added to 2.5 ml of 4% PVA solution and further homogenized
   by ultrasound again to form a water-in-oil-in-water (W/O/W) emulsion.
   The resulting 3.8 ml of W/O/W emulsion was then added to 6 ml of 1% PVA
   solution and stirred for 12 hours at room temperature, allowing the
   organic solvent to evaporate. The nanoparticles were then collected by
   centrifugation at 20,000g for 30 min, resuspended in 1 ml of 4% sucrose
   solution, and lyophilized for 48 hours. Before administration, the
   lyophilized PLGA nanoparticles were reconstituted in endotoxin-free
   PBS.

   The hydrodynamic size of the PLGA nanoparticles was measured by
   Zetasizer Nano ZS (Malvern Instruments). TEM images were acquired using
   an FEI Tecnai G2 F20 Transmission Electron Microscope, using a 200-kV
   acceleration voltage.

Maturation of BMDC in vitro

   As previously described, BMDCs obtained from C57BL/6 mice were
   initially seeded at a density of 1 × 10^6 cells per well in untreated
   round-bottom 24-well plates. Subsequently, 10 μl of PBS, PLGA
   nanoparticles (protein concentration of 4 mg/ml), bacterial lysate
   (protein concentration of 4 mg/ml), or 2 μl of LPS solution (1 mg/ml)
   was added to each well. After 12 hours incubation, cells from each well
   were collected by centrifugation at 1500g for 3 min and resuspended in
   PBS containing 1% FBS. The cells were then stained with anti–CD11c-FITC
   (117306, BioLegend), anti–CD86–phycoerythrin (PE) (159204, BioLegend),
   and anti–CD80-allophycocyanin (APC) (104714, BioLegend) and analyzed
   using flow cytometry on a BD Accuri C6 Plus. The supernatants were
   assessed for cytokine concentrations using TNF-α (SEKM-0034, Beijing
   Solarbio Science & Technology Co. Ltd.), IL-6 (SEKM-0007, Beijing
   Solarbio Science & Technology Co. Ltd.), and IL-12p70 (BMS6004,
   Invitrogen) ELISA kits.

Cellular uptake and Ag presentation in vitro

   DC2.4 cells were seeded at a density of 10^6 cells per well in a
   12-well plate containing coverslips. After 4 hours of incubation, 0.1
   ml of FITC-labeled bacterial protein (0.4 mg/ml), either in its free
   form or encapsulated in PLGA nanoparticles, was added to the
   corresponding wells and incubated for 4, 8, 12, and 24 hours. For
   confocal imaging, cells on each coverslip were fixed with 0.5 ml of 4
   wt % paraformaldehyde solution to preserve cell morphology, followed by
   staining with 1 ml of 75 nM LysoTracker Red and 0.5 ml of DAPI (1
   μg/ml) for 30 min. After staining, the coverslips were thoroughly
   washed and mounted onto glass slides. Imaging was performed using a
   Zeiss LSM 800 confocal laser scanning microscope. For flow cytometry,
   the incubated cells were digested with 0.25% trypsin for 3 min and
   collected by centrifugation at 1200 rpm for 3 min. The cells were then
   washed, and the FITC fluorescence intensity of each group was measured
   using a BD Accuri C6 Plus flow cytometer.

Enrichment of nanoparticles in lymph nodes

   BALB/c mice were intradermally injected with an equivalent amount of
   Cy5.5 (A8103, APExBIO, Houston, USA.)–labeled bacteria proteins,
   encapsulated either in PLGA nanoparticles or in free form. To track the
   fluorescence signals of the bacterial proteins, mice were imaged by
   PerkinElmer IVIS Lumina III imaging system at 4-, 6-, 8-, 24-, and
   36-hour time points postinjection. Following imaging, the mice were
   euthanized and the inguinal lymph nodes were harvested. For confocal
   imaging, the lymph nodes were embedded in paraffin, sectioned, stained
   with DAPI, and observed under a ZEISS LSM 800 confocal microscope. For
   flow cytometry analysis, the lymph nodes were homogenized, filtered
   through a 200-mesh to obtain single-cell suspensions, and divided into
   three groups for staining: group 1: stained with anti–CD11c-FITC
   (117306, BioLegend), anti–CD8a-PE (162304, BioLegend), and
   anti–CD11b-peridinin-chlorophyll-protein complex (PerCP) (101230,
   BioLegend); group 2: stained with anti–CD11c-FITC (117306, BioLegend),
   anti–CD103-APC (110906, BioLegend), and anti–CD11b-PerCP (101230,
   BioLegend); and group 3: stained with anti–CD11c-FITC (117306,
   BioLegend), anti–CD11b-PE (101208, BioLegend), and anti–B220-PerCP
   (103234, BioLegend). All stained cell suspensions were analyzed using a
   BD Accuri C6 Plus flow cytometer.

Immunological evaluation in vivo

   To investigate the immune responses mouse model, inguinal LNs of mice
   with different treatments were collected, homogenized, and filtered
   through a 200-mesh to obtain single-cell suspensions. These suspensions
   were divided into four groups for flow cytometry analysis: group 1:
   stained with anti–CD11c-FITC (117306, BioLegend), anti–CD80-PE (104708,
   BioLegend), and anti–CD86-APC (159216, BioLegend); group 2: stained
   with anti–CD11c-FITC (117306, BioLegend), anti–I-A/I-E-PE (107608,
   BioLegend), and anti–CD40-PerCP (124624, BioLegend); group 3: stained
   with anti–CD3-FITC (100204, BioLegend), anti–CD45-PerCP (103130,
   BioLegend), anti–CD8a-PE (162304, BioLegend), and anti–CD4-APC (100412,
   BioLegend); group 4: stained with anti–CD19-FITC (152404, BioLegend)
   and anti–B220-PerCP (103234, BioLegend). All groups were analyzed using
   a BD Accuri C6 Plus flow cytometer.

   To assess the specific humoral immune response, IgG levels were
   quantified using ELISA. Specifically, lysate of F. nucleatum, S.
   sanguis, E. faecalis, and S. xylosus (protein concentration of 40
   μg/ml) was used to coat the ELISA plates. Ag-specific IgG
   concentrations in the serum were assessed using a series of twofold
   serial dilutions. The horseradish peroxidase goat anti-mouse IgG (H+L)
   antibody (K1221) was purchased from APExBIO, Houston, USA. The endpoint
   titers were presented as the sample dilution, yielding an optical
   density at 450 nm value double the level observed in the control serum.

   To study the specific cellular immune response, mouse spleens were
   collected, homogenized, and filtered through a 200-mesh to obtain
   single-cell suspensions. Red blood cells (RBCs) were lysed using RBC
   lysis buffer, and splenocytes were collected by centrifugation at 1500g
   for 3 min. To assess Ag responsiveness, 1 × 10^6 splenocytes from each
   mouse were co-incubated with 1 μg of bacterial Ag in a 24-well plate
   for 24 hours. After incubation, the splenocytes were collected,
   resuspended in PBS containing 1% FBS, and stained with anti–CD3-FITC
   (100204, BioLegend) and anti–CD8a-PE (162304, BioLegend). The cells
   were then fixed, permeabilized, stained by anti–IFN-γ–APC (505809,
   BioLegend), and analyzed using a BD Accuri C6 Plus flow cytometer.

   To investigate the immune memory response, the number of memory T cells
   in peripheral blood was measured. Blood samples were treated with RBC
   lysis solution, and lymphocytes were collected by centrifugation at
   1500g for 3 min and resuspended in PBS containing 1% FBS. The
   lymphocytes were stained by anti–CD3-FITC (100204, BioLegend),
   anti–CD8a-PerCP (100732, BioLegend), anti–CD44-PE (103007, BioLegend),
   and anti–CD62L-APC (104412, BioLegend) and analyzed using flow
   cytometry on a BD Accuri C6 Plus.

Therapeutic efficiency of PLGA/Ags in inhibiting tumor metastasis

   For the antitumor metastasis experiment, BALB/c mice were injected with
   2.5 × 10^5 4T1-Luc cells infected with F. nucleatum, S. sanguis, E.
   faecalis, and S. xylosus (each at an MOI of 100) on day −1 to establish
   a bacteria-induced breast cancer metastasis model. Subsequently, the
   mice received intradermal injections of 50 μl of a vaccine containing
   Ag (0.2 mg/ml) on days 0 and 6. In monitoring the inhibitory effects of
   PLGA/Ags on metastatic tumor growth, the mice were intraperitoneally
   injected with 0.25 ml of luciferase substrate (15 mg/ml) for
   bioluminescence imaging using the PerkinElmer IVIS Lumina III imaging
   system every 3 days.

   For the prevention of recurrence and metastasis experiment, each BALB/c
   mouse received an intradermal injection of 50 μl of vaccine containing
   Ags (0.2 mg/ml) on days 0, 7, and 14. On day 43, the mice were
   intravenously injected with 2.5 × 10^5 4T1-Luc cells infected with F.
   nucleatum, S. sanguis, E. faecalis, and S. xylosus (each at an MOI of
   100). Every 6 days, the mice were intraperitoneally injected with 0.25
   ml of luciferase substrate (15 mg/ml) for bioluminescence imaging using
   the PerkinElmer IVIS Lumina III imaging system.

Colony-forming unit counting assay

   To evaluate the antibacterial capability, the level of bacteria load in
   the lungs with tumor metastasis was quantified. Specifically, lung
   samples were homogenized, diluted 1:1000, and spread onto Columbia
   blood agar plates. The plates were then incubated at 37°C for 2 days,
   after which the bacterial colonies were counted.

Statistical analysis

   All statistical analyses were evaluated by GraphPad Prism (Prism 8.3.0;
   GraphPad Software, 2019). Two-tailed Student’s t test was used for
   two-group comparisons, and one-way analysis of variance (ANOVA) with a
   Tukey’s post hoc test was used for multiple comparisons. The threshold
   for statistical significance was *P < 0.05, **P < 0.01, ***P < 0.001,
   and ****P < 0.0001. All figure illustrations were created with
   [161]BioRender.com.

Acknowledgments