Probiotics functionalized with a gallium-polyphenol network modulate the intratumor microbiota and promote anti-tumor immune responses in pancreatic cancer

Han, Zi-Yi; Fu, Zhuang-Jiong; Wang, Yu-Zhang; Zhang, Cheng; Chen, Qi-Wen; An, Jia-Xin; Zhang, Xian-Zheng

doi:10.1038/s41467-024-51534-z

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Published: 17 August 2024

Probiotics functionalized with a gallium-polyphenol network modulate the intratumor microbiota and promote anti-tumor immune responses in pancreatic cancer

Nature Communications volume 15, Article number: 7096 (2024) Cite this article

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Abstract

The intratumor microbiome imbalance in pancreatic cancer promotes a tolerogenic immune response and triggers immunotherapy resistance. Here we show that Lactobacillus rhamnosus GG probiotics, outfitted with a gallium-polyphenol network (LGG@Ga-poly), bolster immunotherapy in pancreatic cancer by modulating microbiota-immune interactions. Upon oral administration, LGG@Ga-poly targets pancreatic tumors specifically, and selectively eradicates tumor-promoting Proteobacteria and microbiota-derived lipopolysaccharides through a gallium-facilitated disruption of bacterial iron respiration. This elimination of intratumor microbiota impedes the activation of tumoral Toll-like receptors, thus reducing immunosuppressive PD-L1 and interleukin-1β expression by tumor cells, diminishing immunotolerant myeloid populations, and improving the infiltration of cytotoxic T lymphocytes in tumors. Moreover, LGG@Ga-poly hampers pancreatic tumor growth in both preventive and therapeutic contexts, and amplifies the antitumor efficacy of immune checkpoint blockade in preclinical cancer models in female mice. Overall, we offer evidence that thoughtfully designed biomaterials targeting intratumor microbiota can efficaciously augment immunotherapy for the challenging pancreatic cancer.

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Introduction

Cancer immunotherapy, particularly the treatment modality of immune checkpoint blockade (ICB), has shown remarkable efficacy across various tumor types^1,2,3,4,5. However, its impact on pancreatic ductal carcinoma (PDAC) - a malignancy notorious for its elevated fatality rate - has been noticeably limited⁶. This limitation predominantly arises from the profoundly immunosuppressive dynamics of the tumor microenvironment (TME)^7,8,9. This environment is distinguished by a substantial presence of tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), paired with minimal infiltration of CD8⁺ cytotoxic T lymphocytes (CTLs)^10,11. Further, the tumor cells diminish their immunogenicity by undergoing antigenic loss or mutation, and by frequently attenuating the expression of major histocompatibility complex class I (MCH-I) molecules. This results in an impediment to the recognition and elimination of tumor cells by the immune system^12,13. Moreover, tumor-associated fibroblasts considerably contribute to building physical barriers - primarily through collagen and chemokine secretion - this results in creating a dense extracellular matrix (ECM) that hinders T cell penetration into the core of PDA tumors¹⁴. These collective attributes of the TME advocate for the potential of enhancing PDAC immunotherapy by transitioning the existing immunosuppressive state towards a more immune-activated state. However, current strategies that target these tumor-associated components have made limited progress, underscoring the urgent need for other approaches to augment PDAC immunotherapy¹⁵.

Recent studies have revealed a notable increase in the presence of microbiota in cancerous pancreases compared to healthy ones¹⁶. Furthermore, the diversity of the intratumor microbiome significantly impacts the survival rates of individuals with pancreatic cancer¹⁷. This complex microbial environment interacts with the immune system, promoting immunotolerant pathways within the TME by expanding immunosuppressive immune cells such as TAMs and MDSCs¹⁸. Specifically, the elevated levels of Proteobacteria trigger chronic inflammation and induce PDA tumor cells to release interleukin-1β (IL-1β), which activates dormant pancreatic stellate cells (PSCs) and promotes ECM deposition. This creates an environment of TAM-mediated immunosuppression while impairing the infiltration and functionality of CD8⁺ T cells¹⁹. Moreover, persistent exposure to microbiota-derived lipopolysaccharides (LPS) leads to increased expression of programmed death ligand-1 (PD-L1) in tumor cells, directly compromising the effectiveness of ICB immunotherapy²⁰. In summary, dysregulation of intratumor microbiota in PDAC comprehensively shapes the landscape of tumor immunosuppression by affecting both immune cells and tumor cells. Strategically manipulating the PDA microbiome represents a crucial approach to reshape the tolerogenic innate and adaptive immune TME and enhance the efficacy of checkpoint-based immunotherapy.

In contrast to the extensively studied gut microbiota, the targeted modulation of intratumor microbiota remains largely unexplored²¹. Currently, antibiotics are commonly used to counteract the bacterial community within tumors²². However, routine antibiotic use for this purpose has drawbacks, as broad-spectrum antibiotics may induce bacterial resistance and inadvertently eliminate beneficial microbial communities²³. Reduction in intratumor bacterial α-diversity and richness caused by antibiotics has been associated with shorter-term survival¹⁷. Additionally, the dense ECM of PDA tumors hampers antibiotic penetration, resulting in oral administration alone rarely reaching PDA tumors and potentially causing gut microbiota dysbiosis²⁴. To overcome these limitations and achieve more precise, effective, and safe modulation of the microbiota, other antimicrobials and delivery systems are urgently needed. Gallium, which mimics iron chemically, can disrupt bacterial iron metabolism and exhibit superior antimicrobial effects against various microbes²⁵. Targeting bacterial iron susceptibility with gallium also interferes with multiple iron-related metabolic processes crucial for bacterial survival²⁶. Consequently, the development of resistance to gallium among microbes occurs at a significantly slower rate²⁷. To facilitate the delivery of gallium into PDA tumors^28,29,30, we selected commercial Lactobacillus rhamnosus GG (LGG) which possess the ability to precisely target and infiltrate PDA tumors through the gut-pancreas axis following oral administration³¹. Furthermore, LGG can self-proliferate independently of iron sources, ensuring that gallium does not interfere with its bioactivity³².

Here, we show a strategy utilizing LGG probiotics armed with a gallium-polyphenol network and coated with chitosan nanocoating (LGG@Ga-poly) to modulate the intratumor microbiota and elicit anti-tumor immune response against PDAC (Fig. 1). Oral administration of LGG@Ga-poly selectively targets PDA tumors and effectively eliminates intratumor Proteobacteria and microbiome-derived LPS by inhibiting bacterial iron metabolism mediated by Ga³⁺. This process inhibites the activation of Toll-like receptors (TLRs) dependent on microbial presence, leading to a reversal of the immunosuppressive TME. It results in a reduction in immunotolerant myeloid populations and enhanced recruitment of T cells. Notably, LGG@Ga-poly demonstrates significant delay in tumor initiation in a preventive setting and shows potential as an independent therapy or in combination with checkpoint-based immunotherapy for established PDA tumors. This biohybrid material effectively and safely modulates the intratumor microbiome, enhancing the overall efficacy of ICB immunotherapy for challenging PDAC³³.

**Fig. 1: Schematic illustration of LGG@Ga-poly for PDA immune activation through modulation of intratumor microbiota.**

Results

Intratumor microbiota dysbiosis induces PDAC immunosuppression

Firstly, we evaluated the therapeutic effectiveness of ICB in subcutaneous and orthotopic murine models using Panc-02 cells. We observed that anti-PD-L1 (α-PD-L1) treatment significantly suppressed tumor growth in subcutaneous tumors but showed no inhibition in orthotopic tumors (Fig. 2a, b). Immunofluorescence staining revealed a higher population of T cells in subcutaneous tumors compared to orthotopic tumors (Supplementary Fig. 1a). The distinct response to ICB was unlikely due to genetic factors as the cell line and mouse strain were identical³⁴. Recognizing the importance of host-microbe interactions in immune modulation³⁵, we hypothesized that intratumor microbes might be responsible for inducing resistance to PDAC immunotherapy. To test this hypothesis, we analyzed the profiles of intratumor microbiota in the two tumor models. Fluorescence in situ hybridization (FISH) assays confirmed a substantial presence of microbes in orthotopic tumors, in contrast to subcutaneous tumors (Supplementary Fig. 1b). Furthermore, elevated levels of microbiota-derived LPS were detected in orthotopic tumors, while minimal LPS was observed in subcutaneous tumors (Fig. 2c).

**Fig. 2: Intratumor microbiota induced PDA TME immunosuppression and restrained α-PD-L1 immunotherapy efficacy.**

To confirm the contribution of intratumor microbiota to the unresponsiveness of PDA to α-PD-L1 therapy, we investigated the potential of antibiotics to restore responsiveness to checkpoint-based immunotherapy. Initially, we compared the microbiota profiles between normal and cancerous pancreases using 16 S rDNA gene sequencing³⁶. The analysis revealed a significant increase in Proteobacteria and a decrease in Firmicutes in PDA tumors compared to normal pancreases (Fig. 2d and Supplementary Fig. 2a). Moreover, the intratumor content of LPS was significantly higher in PDA tumors than in normal pancreases (Fig. 2e). When combined with polymyxin B (PmB)³⁷, an antimicrobial peptide that inactivates LPS-expressing bacteria, the antitumor efficacy of α-PD-L1 treatment improved significantly (Fig. 2f). The combination therapy of PmB and α-PD-L1 resulted in durable tumor regression and increased infiltration of T cells (Fig. 2f, g). Correspondingly, there was a notable reduction in bacterial load in the antibiotic-treated group, accompanied by a significant decrease in LPS levels (Fig. 2h and Supplementary Fig. 2b). The LSL-KrasG12D-Pdx-1-Cre (KPC) transgenic mouse model closely replicates many features of human pancreatic cancer, and we found that antibiotic treatment in this model also significantly enhanced the efficacy of α-PD-L1 (Supplementary Fig. 3a).

To gain a more comprehensive understanding of the role of intratumor microbiota in immune suppression in PDAC, we conducted experiments to investigate whether microbiota within the TME triggers immunosuppression through the production of LPS, a metabolite derived from the microbiota. We co-inoculated LPS and tumor cells directly into the pancreas of mice and collected PDA tumors after four weeks for histological analysis. Hematoxylin-eosin (H&E) staining revealed that LPS promoted tumor growth, while immunofluorescence staining indicated an increase in intratumor α-smooth muscle actin (α-SMA) and Collagen I content³⁸, suggesting heightened inflammation within the tumor (Supplementary Fig. 3b, c). Additionally, we collected immune cells from tumors and lymph nodes for flow cytometry (FCM) analysis. The presence of LPS led to a significant decrease in antigen-presenting cells (APCs), including mature dendritic cells (DCs) and M1-like macrophages with antitumor properties. Notably, there was a notable increase in the proportions of M2-like macrophages and MDSCs after LPS treatment. Furthermore, the percentage of cytotoxic CD8⁺ T cells, which play a crucial role in antitumor immunity, was reduced in the LPS-treated group (Fig. 2i). In vitro assays demonstrated that LPS did not directly affect the growth and migration of PDA tumor cells (Supplementary Fig. 4). Overall, these findings provide evidence that intratumor microbiota, particularly microbial LPS, mediates tumor immunosuppression in PDAC and contributes to resistance against ICB immunotherapy.

Preparation and characterization of LGG@Ga-poly

To eliminate intratumor microbiota, we utilized LGG probiotics to promote the targeting and penetration of bacteria-inactivated Ga³⁺ into the tumors (Fig. 3a). After screening various antibacterial metal ions, we identified Ga³⁺ as an effective agent for inactivating multiple microbes while causing minimal toxicity to LGG (Fig. 3b and Supplementary Fig. 5). Functionally, Ga³⁺ eliminates microbes by mimicking iron ions and disrupting iron-related metabolic pathways within bacteria. Interestingly, the metabolism of LGG is not dependent on iron, rendering it resistant to inactivation by Ga³⁺ (Supplementary Fig. 6)³⁹. Next, we coated LGG with a biocompatible supramolecular coating composed of Ga³⁺ and plant polyphenols. We screened three kinds of polyphenols⁴⁰, including tannin (TA), gallic acid (GA), and epigallocatechin gallate (EGCG), and found that the antibacterial activity of Ga³⁺ for Escherichia coli was enhanced when coordinated with polyphenols (Fig. 3c). The bacterial carrier for iron uptake, known as siderophore⁴¹, is a polyphenolic structure, suggesting that the gallium-polyphenol coordination can improve Ga³⁺ endocytosis within bacterial cells. This hypothesis was confirmed by inductively coupled plasma (ICP) measurement, with more Ga³⁺ being detected in bacteria in the presence of polyphenols (Supplementary Fig. 7). When Ga³⁺ was combined with EGCG, the most bacterial death was observed. Therefore, we self-assembled a gallium-EGCG network as a single-cell nanocoating to encapsulate probiotic cells. To prevent the premature leakage of Ga³⁺ in the gastrointestinal (GI) tract, we further deposited positively charged chitosan films by electrostatic cross-linking to form LGG@Ga-poly.

**Fig. 3: Preparation and in vitro antimicrobial behavior of LGG@Ga-poly.**

The changes in zeta potentials of LGG confirmed the successful deposition of each layer (Fig. 3d). Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations indicated that LGG@Ga-poly displayed a distinct polymer layer around bacterial cells (Supplementary Fig. 8). TEM-assisted element mapping of C, O, and Ga elements confirmed that the Ga-based polymer layers were located on the bacterial cell surface (Fig. 3e). After Ga-poly deposition, the solutions darkened, and correspondingly, the UV-visible spectrum was changed (Supplementary Fig. 9). Furthermore, Ga-EGCG and chitosan did not inhibit LGG growth and bioactivity (Fig. 3f). We evaluated the tolerance capacity of LGG@Ga-poly in different conditions, including simulated gastric fluid (SGF) and simulated colonic fluid (SCF). The viability of LGG@Ga-poly was not significantly affected after treatment with SGF and SCF, as shown in Fig. 3g and Supplementary Fig. 10a. The loading efficacy of Ga³⁺ was determined as 56.7%, and the chitosan deposition effectively prevented Ga³⁺ leakage in the SGF (Fig. 3h). This feature is beneficial in reducing fluctuations in the gut microbiota, thereby enhancing biosafety.

Next, we evaluated the antibacterial and LPS-scavenging abilities of LGG@Ga-poly. LGG alone exhibited a slight inhibitory effect on bacterial growth due to nutrient competition and metabolic antagonism. In contrast, LGG@Ga-poly effectively inhibited the growth of bacteria such as E. coli, Staphylococcus aureus (S. aureus), and cancer-promoting Fusobacterium nucleatum (F. nucleatum), which are commonly found in PDA tumors (Fig. 3i and Supplementary Fig. 10b, c). Bacterial plate counting further confirmed the superior antimicrobial behavior of LGG@Ga-poly (Supplementary Fig. 10d). Then, we constructed a F. nucleatum-infected Panc-02 cell model to investigate the antibacterial ability of materials for intracellular bacteria, and the results showed that both Ga³⁺ and LGG@Ga-poly could inhibit intracellular bacterial growth (Supplementary Fig. 10e). The addition of exogenous Fe³⁺ significantly inhibited the antibacterial effect of LGG@Ga-poly, indicating that LGG@Ga-poly kills bacteria by disrupting bacterial iron metabolism (Fig. 3j).

The dense ECM in PDA tumors hinders the penetration of free antimicrobials, resulting in their retention in the intestines. This can disrupt the balance of gut microbiota and lead to unintended side effects. To investigate whether LGG@Ga-poly can overcome these biological barriers in PDA tumors, we established a 3D tumor sphere infected with E. coli for in vitro experiments. As shown in Supplementary Fig. 11a, free Cy5 was only observed in the outermost layer of the tumor sphere, while LGG modified with Cy5 (LGG@Cy5) effectively penetrated the entire tumor sphere. Leveraging the natural active mobility of bacteria and their hypoxia-tropism behavior, LGG@Cy5 reached deeper, hypoxic tumor regions. Furthermore, LGG@Ga-poly delivered more Ga³⁺ to the tumor spheres compared to the group treated with free Ga³⁺ (Supplementary Fig. 11b). LGG@Ga-poly also exhibited enhanced penetration in the E. coli-infected tumor spheres (Fig. 3k). As expected, Ga³⁺ alone did not significantly inhibit bacterial growth, while LGG@Ga-poly effectively eliminated both microbes and LPS in the tumor spheres (Fig. 3l).

In vivo modulation of intratumor microbiota by LGG@Ga-poly

Initially, we assessed the biodistribution of LGG@Ga-poly in an orthotopic Panc-02-bearing mouse model following oral administration. LGG@Ga-poly, labeled with Cy5.5, was tracked using the in vivo imaging system (IVIS) for whole-body fluorescence imaging of mice. Fluorescence signals were observed in tumor areas as early as 2 hours post-administration, reaching a peak at 8 hours and maintaining strong intensity even after 7 days. Ex vivo fluorescence imaging of tumors and major organs further confirmed the promising tumor-targeting capability of LGG@Ga-poly (Fig. 4a and Supplementary Fig. 12a). Histological imaging demonstrated specific accumulation of LGG@Ga-poly within tumor tissues, while minimal accumulation was observed in normal organs (Supplementary Fig. 12b). Moreover, the concentration of Ga³⁺ in tumors was higher compared to major organs (Fig. 4b). Photoacoustic imaging (PAI) revealed that tumors treated with [email protected] exhibited a more pronounced photoacoustic signal compared to tumors treated with free Cy5.5, indicating enhanced tumor penetration of the antimicrobials facilitated by the LGG (Fig. 4c).

**Fig. 4: Modulation of intratumor microbiota by LGG@Ga-poly.**

Subsequently, we evaluated the in vivo modulation of intratumor microbiota by LGG@Ga-poly. At the end of the treatment, orthotopic tumors were collected for analysis of intratumor microbiota. No significant difference in operational taxonomic units (OTUs) was observed between the control and LGG@Ga-poly treatment groups (Fig. 4d). The α-diversity of intratumor microbiota, as measured by the Simpson diversity index, remained unchanged (Fig. 4e). However, LGG@Ga-poly treatment induced a distinct clustering of the microbial community structure, as indicated by principal components analysis (PCA) reflecting β-diversity (Fig. 4f). Analysis at the phylum level demonstrated that LGG@Ga-poly treatment significantly reduced the abundance of Proteobacteria, a group of bacteria associated with pro-tumoral effects (Fig. 4g and Supplementary Fig. 13). At the genus level, Lactobacillus richness was notably enhanced following LGG@Ga-poly treatment, suggesting successful niche competition (Fig. 4h). Furthermore, the overall bacterial load within tumors decreased after LGG@Ga-poly treatment (Fig. 4i). Correspondingly, the concentration of LPS within tumors treated with LGG@Ga-poly was approximately 5-fold lower compared to tumors treated with PBS (Fig. 4j).

A compromised intestinal barrier can lead to the infiltration of bacterial LPS from the gut into the bloodstream and tumor tissues, exacerbating tumor immunosuppression. To address this concern, we thoroughly evaluated the impact of LGG@Ga-poly treatment on both gut microbiota and intestinal barrier function. Our assessment revealed that oral administration of LGG@Ga-poly minimally affected gut microbiota homeostasis, as evidenced by no significant changes in bacterial OTUs, α-diversity, or phylum-level diversity (Supplementary Fig. 14). Additionally, the concentration of LPS in the gut did not show any significant changes following different treatments (Fig. 4k). To assess intestinal barrier integrity, an in vivo intestinal permeability assay using FITC-dextran was conducted⁴². The results demonstrated a remarkable 2-fold decrease in FITC concentration in the blood after treatment with LGG@Ga-poly, indicating the restoration of intestinal barrier function (Fig. 4l). Consequently, LGG@Ga-poly treatment resulted in a significant threefold decrease in detected blood levels of LPS compared to the PBS-treated group (Fig. 4m). In conclusion, we have demonstrated that oral administration of LGG@Ga-poly effectively eliminates intratumor microbiota and microbiota-derived LPS, while concurrently restoring intestinal homeostasis to mitigate LPS leakage into the bloodstream (Fig. 4n).

In vivo antitumor responses

Firstly, we evaluated the therapeutic effect of different materials in a prophylactic setting. Healthy C57BL/6 mice were pretreated with different materials, including PBS, LGG, Ga-poly, LGG + Ga-poly (physical mixture of LGG and Ga-poly), and LGG@Ga-poly, followed by in situ injection of Panc-02 cells into the pancreas (Fig. 5a). Three weeks later, mice were sacrificed and tumors were collected. The smallest tumor volume and the lightest tumor weight in the LGG@Ga-poly group indicated that LGG@Ga-poly could effectively delay tumorigenesis (Fig. 5b, c). The terminal deoxynucleotidyl transferase 2’-deoxyuridine, 5’-triphosphate (dUTP) nick end labeling (TUNEL) assay showed that LGG@Ga-poly induced the highest number of apoptotic tumor cells (Fig. 5d). Additionally, CD3-immunofluorescence staining of tumors indicated that LGG@Ga-poly potentiated antitumor T cell responses (Fig. 5e). The results obtained using the KPC mouse model also demonstrated that LGG@Ga-poly significantly inhibited the formation and growth of tumors (Supplementary Fig. 15a). Furthermore, when LGG@Ga-poly was administered orally to healthy mice over a three-month period, we observed no significant changes in immune-related indicators, indicating that long-term prevention with LGG@Ga-poly does not adversely affect the immune system (Supplementary Fig. 15b). Collectively, these findings suggest that LGG@Ga-poly significantly reduces PDA tumorigenesis, offering a potential preventative benefit for individuals at risk of pancreatic cancer.

We next evaluated the therapeutic efficacy of LGG@Ga-poly in a therapeutic setting. Orthotopic Panc-02-bearing mice were randomly divided into five groups, and all treatments were orally administered every two days for a total of eleven times (Fig. 5f). Real-time tumor growth was monitored using the IVIS system by measuring the bioluminescence of cancer cells. Mice treated with LGG@Ga-poly exhibited the weakest bioluminescence intensity, indicating the smallest tumor size (Supplementary Fig. 16a). Tumor growth was also monitored using magnetic resonance imaging (MRI) and μ-computerized tomography (μ-CT) imaging, where mice in the PBS-treated group showed obvious suspected areas, while almost no tumors were observed after LGG@Ga-poly treatment (Fig. 5g, h). The observation of resected tumors and tumor weighing also indicated the superior therapeutic efficacy of LGG@Ga-poly (Fig. 5i, j). Additionally, LGG@Ga-poly treatment induced the largest tumor necrosis regions and tumor cell apoptosis, as demonstrated by H&E and TUNEL staining, and significantly inhibited tumor cell proliferation, as shown by Ki67 staining (Fig. 5k). Bacterial Gram staining of tumors demonstrated that LGG@Ga-poly remarkably reduced the overall loads of LPS-expressing Gram-negative microbes, and Masson staining showed that LGG@Ga-poly-treated tumors had the least ECM deposition (Supplementary Figs. 16b, c). Furthermore, orally administered LGG@Ga-poly remarkably inhibited the lung metastasis of PDA tumors (Fig. 5l).

The safety profiles after LGG@Ga-poly treatment were then carefully evaluated in vivo. As all components used in the system, including LGG, chitosan, Ga³⁺, and EGCG, are classified as Generally Recognized as Safe (GRAS) and widely employed in medical practices, LGG@Ga-poly exhibited excellent biocompatibility and clinical translatability. Throughout the entire treatment period, there were only minor fluctuations in the body weight of mice, indicating no significant toxicity (Fig. 5m). H&E staining of major organs showed no visible abnormalities induced by LGG@Ga-poly. Blood biochemistry and blood routine tests further indicated the biosafety of LGG@Ga-poly treatment (Supplementary Fig. 17a, b).

We also investigated cancer stem cells across various treatment groups and found that the material treatments did not alter the proportion of cancer stem cells. Importantly, the percentage of cancer stem cells in the tumor mass of the LGG@Ga-poly-treated group was similar to that observed in the PBS-treated mice (Supplementary Figs. 17c–e). Additionally, we evaluated the antitumor efficacy of LGG@Ga-poly in the KPC mouse model. Six-week-old KPC mice were orally administered each material every two days for a total of twenty doses. After treatment, the mice were sacrificed, and the tumors were weighed. The results showed that LGG@Ga-poly treatment achieved the highest therapeutic efficacy (Supplementary Fig. 18).

Interplay between microbiota modulation and systemic antitumor immune responses

To investigate the impact of microbiota elimination on the immune TME, we assessed immune cell infiltration using FCM after administering various treatments. The results in Supplementary Fig. 19 showed that after LGG@Ga-poly treatment, there was a significant increase in the proportion of APCs, including mature DCs and M1-like macrophages (Fig. 6a, b). The expression of MHCII on monocytes was also increased (Fig. 6c). In contrast, the number of M2-like macrophages and MDSCs associated with tumor immunosuppression was significantly decreased following LGG@Ga-poly treatment (Fig. 6d, e). Furthermore, LGG@Ga-poly treatment led to the highest infiltration of CD8⁺ T cells within tumor tissues and significantly reduced the presence of immunosuppressive regulatory T cells (Tregs) (Fig. 6f, g). To further evaluate the functional changes in these CD8⁺ T cells, we detected the percentage of CD8⁺ T cells that secreted IFN-γ and granzyme B (GzmB). Remarkably, the levels of both IFN-γ and GzmB-positive CD8⁺ T cells were substantially increased following treatment with LGG@Ga-poly (Fig. 6h, i). Additionally, the LGG@Ga-poly-treated group exhibited a significant increase in effector memory T (T_EM) cells in the spleen compared to other groups (Supplementary Fig. 20a). Notably, PD-L1 expression by tumor tissues also significantly decreased after LGG@Ga-poly treatment (Supplementary Fig. 20b). A comprehensive analysis of immune-related cytokines within tumor tissues revealed that LGG@Ga-poly treatment led to a significant upregulation of antitumor cytokines, including IL-6, IL-12, IFN-γ, and TNFα, and a marked downregulation of immunosuppressive cytokines, such as IL-10 and IL-1β, as shown in Fig. 6j and Supplementary Fig. 20c. These findings suggest LGG@Ga-poly effectively modulates the immune TME to promote an anti-tumor immune response.

**Fig. 6: LGG@Ga-poly treatment induced an immune-active PDA TME.**

To further assess the immune response within tumor tissues following treatment with LGG@Ga-poly, we conducted a transcriptomic analysis of 14,518 genes within the tumors. A Venn diagram was used to illustrate the gene expression relationship between the control and LGG@Ga-poly treatment group. Using a threshold of fold change ≥2 and p < 0.05, 1265 differentially expressed genes were identified (Fig. 7a), and a volcano plot was used to illustrate the differences in gene expression between the two groups (Fig. 7b). Genes associated with antitumor immunity were further analyzed by gene ontology (GO) function enrichment analysis, which demonstrated a significant enrichment in “regulation of innate immune response”, “positive regulation of immune effector process”, and “positive regulation of lymphocyte activation” after LGG@Ga-poly treatment (Fig. 7c). Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis revealed that the immune response activated by LGG@Ga-poly primarily relied on the “Toll-like receptor (TLR) signaling pathway”, “NOD-like receptor signaling pathway”, and “NF-kappa B signaling pathway” (Fig. 7d). These above results demonstrate that the innate immunity, especially the TLR signaling pathway is of significance on immune-activation by LGG@Ga-poly.

**Fig. 7: Mechanism analysis of LGG@Ga-poly for PDA immune activation.**

Following this, a comprehensive approach involving database analysis, in vitro assays, and in vivo experiments was undertaken to validate the capacity of LGG@Ga-poly to counteract PDA immunosuppression through the modulation of the LPS/TLRs signaling pathway. Our analysis of the TCGA database unveiled a substantial increase in the expression levels of TLRs in PDA tumors when compared to healthy pancreatic tissues (Fig. 7e, and Supplementary Fig. 21a)⁴³. Notably, the expression of TLR4 exhibited an inverse correlation with patient survival in pancreatic cancer, underscoring its potential adverse impact on patient outcomes (Supplementary Fig. 21b). Furthermore, a positive association was observed between TLR4 expression and CD274 (PD-L1) expression, suggesting the conceivable presence of an immunosuppressive effect linked to TLR4 activation (Supplementary Fig. 21c). Subsequent investigations encompassed an in vitro assay aimed at elucidating the impact of LPS stimulation on TLR4 expressed by macrophages. The short-term stimulation of LPS prompted the polarization of RAW264.7 macrophages towards the M1 phenotype; however, prolonged exposure to LPS for over 72 hours significantly elevated the proportion of immunologically tolerant M2-like macrophages (Fig. 7f, and Supplementary Fig. 21d, e). These findings indicate that extended LPS exposure within PDA tumors can instigate an immunosuppressive TME. Complementing this with in vivo evidence, we observed a significant reduction in TLR expression within tumor tissues upon LGG@Ga-poly treatment. Besides, when combined with LPS injection to sustain TLR/NF-kappa B signaling activation, the immune-activating effect of LGG@Ga-poly was notably curtailed (Fig. 7g, h, and Supplementary Fig. 22). Correspondingly, discernible inhibition of tumor growth was not observed (Fig. 7i). Collectively, our findings elucidate that LGG@Ga-poly treatment effectively abrogates PDA immunosuppression by inhibiting microbial-dependent TLR activation within the TME.

Synergistic effect with chemotherapy and immunotherapy

Finally, we evaluated the synergistic effect of LGG@Ga-poly treatment with gemcitabine (Gem) chemotherapy and ICB immunotherapy. Previous studies have shown that modulating the intratumor microbiota can enhance the efficacy of chemotherapy in PDAC^44,45. To assess the enhanced therapeutic effect of LGG@Ga-poly, we randomly assigned Panc-02-bearing mice into four groups: PBS, Gem, LGG@Ga-poly, and LGG@Ga-poly + Gem. LGG@Ga-poly was administered orally, while Gem was injected intravenously (Fig. 8a). Throughout the treatment period, the body weight of mice treated with LGG@Ga-poly + Gem fluctuated slightly, while Gem treatment alone led to a noticeable decline in mice weight, suggesting that the combination of Gem with LGG@Ga-poly could alleviate chemotherapy side effects (Fig. 8b). At the end of the treatment, mice were sacrificed, and tumors were dissected for weighing, which revealed that the combined therapy effectively suppressed tumor growth by approximately 80% compared to the PBS control, while Gem alone only inhibited tumor growth by about 15% (Fig. 8c). H&E staining and TUNEL assay indicated that LGG@Ga-poly combined with Gem induced the highest amount of apoptotic tumor cells, while showed no obvious toxocity on normal organs (Fig. 8d and Supplementary Fig. 23a). Immunofluorescence staining for CD3 demonstrated that LGG@Ga-poly plus Gem treatment potentiated T cell infiltration to the maximum extent possible (Fig. 8e). Blood biochemical and blood routine tests confirmed the in vivo safety of combination chemotherapy (Supplementary Fig. 23b). The level of the probiotic Butyricicoccus in the gut microbiome improved following treatment with LGG@Ga-poly plus chemotherapy, compared to the free Gem group. This suggests that LGG@Ga-poly plays a role in stabilizing the gut microbiome (Supplementary Fig. 23c).

**Fig. 8: Synergistic antitumor effect with chemotherapy and α-PD-L1 therapy.**

Furthermore, we confirmed that LGG@Ga-poly treatment could enhance the therapeutic efficacy of α-PD-L1 immunotherapy. The therapeutic regimen is shown in Fig. 8f. The combination of LGG@Ga-poly and α-PD-L1 mostly inhibited tumor growth (Fig. 8g, h). Importantly, mice treated with the combined therapy survived beyond 60 days, while those treated with free α-PD-L1 all died within 45 days (Fig. 8i). Mice weight in each group was slightly changed (Fig. 8j). Additionally, we observed improved intratumor infiltration of CD8⁺ T cells with the combined LGG@Ga-poly and α-PD-L1 therapy (Fig. 8k). Moreover, H&E staining of normal organs indicated that the combined ICB therapy did not induce metastasis niches (Supplementary Fig. 24). These results indicate that LGG@Ga-poly has the potential to improve the effectiveness of PDAC treatment by further combining chemotherapy and ICB-based immunotherapy.

Discussion

This study highlights the crucial role of intratumor microbiota, specifically microbiota-derived LPS, in promoting immunosuppression and resistance to ICB treatment in PDAC. To address this, we have developed a biohybrid system called LGG@Ga-poly as an oral biotherapeutic for enhancing PDAC immunotherapy by modulating the microbiota-immune interactions. Orally administered LGG@Ga-poly selectively targets PDA tumors via gut-pancreas translocation and effectively eliminates intratumor microbiota and LPS via the biological competition of LGG and Ga³⁺-mediated microbial inactivation. This process disrupts the immunosuppressive TME by reducing TAMs and MDSCs, while increasing cytotoxic CD8⁺ T cells. The immune-activation mechanism is attributed to the inhibition of microbial-dependent activation of the TLR4/NF-kappa B signaling pathway. Moreover, LGG@Ga-poly significantly delays PDA tumorigenesis in a preventive setting, and inhibits established tumor growth and lung metastasis, and enhances the efficacy of ICB immunotherapy. These findings provide evidence that biomaterials can be rationally designed to modulate intratumor microbiota for improving PDAC immunotherapy.

Our design offers several notable advantages. Firstly, the bactericidal action induced by Ga³⁺ relies on iron availability, which is not required by LGG, ensuring that Ga³⁺ has no adverse effect on LGG viability. This characteristic makes LGG@Ga-poly highly stable and bioactive. Additionally, the exceptional tumor-targeting capability, coupled with chitosan protection, prevents premature release of free Ga³⁺ in the gastrointestinal tract, minimizing disruptions to gut microbiota equilibrium. Furthermore, all components of LGG@Ga-poly are GRAS, demonstrating excellent biocompatibility and potential for clinical translation. In conclusion, this research paves the way for the development of highly targeted living materials that can modulate the tumor microbiome, thereby enhancing the effectiveness of cancer immunotherapy for various types of microbe-colonized cancer.

Methods

This research complies with all relevant ethical regulations approved by the Institutional Animal Care and Use Committee (IACUC) of the Animal Experiment Center at Wuhan University, China. All mice were housed at 12-h light-dark cycle within 25-27 °C and 45-55% humidity. In our work, no subcutaneous tumor burden exceeds 2000 mm³, which meets the maximum tumor burden permitted by the protocol of the Animal Care and Use Committee of Wuhan University. Tumor volume was calculated as: width²×length/2. For orthotopic tumor-bearing mice, mice were euthanized when the animals became moribund, exhibiting severe weight loss, extreme weakness, or unhealing ulceration. All experimental mice were female. The study design did not account for gender differences, given their minimal impact on the influence of intratumor microbiota on pancreatic cancer.

Materials

Ga(NO₃)₃·6H₂O (CAS: 69365-72-6), tannin (TA, CAS: 1401-55-4), gallic acid (GA, CAS: 149-91-7), epigallocatechin gallate (EGCG, CAS: 989-51-5), and chitosan (CS, CAS: 9012-76-4) were purchased from Sinopharm Group Co. Ltd. Cyanine 5.5 (Cy5.5, CAS: 146368-15-2) was purchased from Beyotime Institute of Biotechnology. FITC-dextran (CAS: 60842-46-8) and LPS (CAS: 297-473-0) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Luria-Bertani (LB) medium, M9 liquid medium, Brain-Heart Infusion Broth (BHI) medium, and MRS Broth medium were purchased from Guangdong Huankai Microbial SCI & Tech Co., Ltd. Cell culture dishes/plates and 20-mm glass-bottom dishes were purchased from NEST Biotechnology co., ltd. Dulbecco’s Modified Eagle Medium (DMEM) was acquired form Sperikon Life Science & Biotechnology co., ltd. Fetal bovine serum (FBS), penicillin, streptomycin, and trypsin were obtained from VivaCell (Shanghai, China). Transwell chamber was purchased from Zhejiang Saining Biotechnology Co., Ltd. Dimethylthiazol-2-yl]−2,5-diphenyltetrazoliumbromide (MTT), were obtained from GIBCO Invitrogen Corp.

Antibodies

The antibodies used for FCM were as follows: anti-mouse CD3 (FITC, Catalog number: 100203, Clone: 17A2, 1:50 dilution), anti-mouse CD4 (APC, Catalog number: 100411, Clone: GK1.5, 1:100 dilution), anti-mouse CD8a (PE, Catalog number: 100707, Clone: 53-6.7, 1:100 dilution), anti-mouse CD11c (FITC, Catalog number: 117305, Clone: N418, 1:200 dilution), anti-mouse CD80 (PE, Catalog number: 104707, Clone: 16-10A1, 1:50 dilution), anti-mouse CD86 (APC, Catalog number: 105011, Clone: GL1, 1:100 dilution), anti-mouse CD11b (APC, Catalog number: 101211, Clone: M1/70, 1:100 dilution), anti-mouse Ly-6G (Gr-1) (FITC, Catalog number: 108405, Clone: RB6-8C5, 1:200 dilution), anti-mouse F4/80 (FITC, Catalog number: 123107, Clone: BM8, 1:200 dilution), anti-mouse CD206 (PE, Catalog number: 141705, Clone: C068C2, 1:50 dilution), anti-mouse CD44 (PE, Catalog number: 103023, Clone: IM7, 1:100 dilution), anti-mouse CD62L (APC, Catalog number: 104411, Clone: MEL-14, 1:100 dilution), anti-mouse CD4 (PE, Catalog number: 116006, Clone: RM4-4, 1:100 dilution), anti-mouse CD8a (APC, Catalog number: 100711, Clone: 53-6.7, 1:100 dilution), anti-mouse Foxp3 (PE, Catalog number: 126403, Clone: MF-14, 1:20 dilution), anti-mouse IFNγ (PE, Catalog number: 163503, Clone: W18272D, 1:200 dilution), anti-mouse GzmB (PE, Catalog number: 372207, Clone: QA16A02, 1:20 dilution). All antibodies used for FCM and anti-PD-L1 antibody were purchased from BioLegend.

Characterization

SEM images were obtained on Zeiss Sigma FESEM. TEM images were obtained by a JEM-2100 (JEOL) transmission electron microscope. Zeta potential was measured on a Nano-ZS ZEN3600 particle sizer (Malvern Instruments). UV-vis absorbance was measured by UV-vis spectroscopy (Lambda Bio40). Confocal microscopy images were obtained from a confocal laser scanning microscope (CLSM, Nikon C1-si TE2000). Cell viability was evaluated through the MTT assay using a microplate reader (Bio-Rad, Model 550, USA). Blood routine was measured by an Auto Hematology Analyzer (MC-6200VET), and blood biochemistry was analyzed by a biochemical auto analyzer (MNCHIP, Tianjin, China). In vivo imaging was performed by a Spectrum Pre-clinical In vivo Imaging System (Pekin Emer). μ-CT scanning was conducted on a Quantum FX microCT system (PerkinElmer). MRI imaging was obtained from an Aspect M3 MRI imaging system (Israel). PAI imaging was conducted by a Vevo® LAZR system (visualSonics, Canada). Immune cell fluorescence intensity was analyzed by flow cytometry (BD FACS Aria III, USA).

Bacteria culture

E. coli and S. aureus were grown aerobically in LB medium consisting of 10 mg mL⁻¹ tryptone, 5 mg mL⁻¹ yeast extract, and 0.5 mg mL⁻¹ NaCl, overnight at 37 °C. B. subtilis was grown aerobically in BHI medium containing 17.5 mg mL⁻¹ brain heart immersion powder, 2 mg mL⁻¹ D-glucose, 10 mg mL⁻¹ tryptone, 5 mg mL⁻¹ NaCl, and 2.5 mg mL⁻¹ NaH₂PO₄, at 37 °C for 24 hours. F. nucleatum was anaerobically grown in BHI medium at 37 °C for 24 hours. LGG was anaerobically grown in Modified MRS medium consisting of 10 mg mL⁻¹ proteose peptone, 5 mg mL⁻¹ beef extract, 4 mg mL⁻¹ yeast extract, 20 mg mL⁻¹ dextrose, 1 mg mL⁻¹ polysorbate 80, 2 mg mL⁻¹ ammonium citrate, 5 mg mL⁻¹ sodium acetate, 2 mg mL⁻¹ dipotassium phosphate, 0.1 mg mL⁻¹ magnesium sulfate, and 0.05 mg mL⁻¹ manganese sulfate, at 37 °C for 24 hours. All bacterial lines were purchased from China Center for Type Culture Collection (CCTCC).

Cell culture

The human pancreatic cancer cell line Panc-01 (Catalog no. GDC0309), human pancreatic stellate cell line HPSC (Catalog no. GPC0058), and RAW264.7 (Catalog no. GDC0143) cell lines were obtained from CCTCC. The mouse pancreatic cancer cell line Panc-02 (CL-0736) were kindly provided by Wuhan Pricella Biotechnology Co., Ltd. Panc-02, Panc-01, and HPSC were cultured in a humidified atmosphere with 5% CO₂ using DMEM supplemented with 10% FBS (v/v) and 1% antibiotics (penicillin-streptomycin, 10,000 U mL⁻¹) at 37 °C. The Panc-02^luc cell line, which was transfected with luciferase, was cultured in DMEM containing 5% puromycin to maintain the selection pressure for the transfected cells. The RAW264.7 macrophage cell line was cultured in DMEM supplemented with heat-inactivated FBS. Panc-02 cell line was authenticated by short tandem repeat (STR) profiling, and other cell lines we used were morphologically confirmed according to the information provided by CCTCC.

Murine tumor models

Six-week-old C57BL/6 female mice (18 ± 2 g) were obtained from the Animal Biosafety Level III Lab at Wuhan University. All animal experimental procedures were conducted in accordance with the guidelines set by the Institutional Animal Care and Use Committee (IACUC) of the Animal Experiment Center at Wuhan University, China. To establish a subcutaneous PDAC mouse model, each mouse was injected with 100 μL of Panc-02 cells (1 × 10⁶ cells per mouse) into the right flank. The mice were then monitored for 7 days to allow tumor development. For the establishment of an orthotopic PDAC mouse model, 50 μL of luciferase-transfected Panc-02^luc cells (1 × 10⁶ cells per mouse) were inoculated into the pancreas of each mouse. After transplantation, the mice were observed for 7 days to allow tumor growth.

In vivo antitumor efficacy of α-PD-L1

The Panc-02-bearing mice were randomly assigned to two groups in subcutaneous or orthotopic tumor-bearing mouse models (nine mice in each group). The treatment group received intraperitoneal injections of 100 μL of 0.6 mg kg⁻¹ α-PD-L1 on the 3^rd, 6^th, and 9^th day, while the control group was treated with 100 μL of PBS. Tumor volume was measured every other day for a total of eighteen days. In the orthotopic Panc-02-bearing mouse model, tumor volume was assessed by monitoring the bioluminescence of cancer cells using the IVIS system.

In vivo antitumor efficacy of antibiotics with α-PD-L1

Orthotopic Panc-02-bearing PDAC mice were randomly assigned to two groups (six mice in each group). One group was orally administered polymyxin B (50 mg kg⁻¹) on the 3^rd, 6^th, and 9^th day, while the other group received 100 μL of PBS. On the 3^rd, 6^th, and 9^th day, both groups were intraperitoneally injected with 100 μL of 0.6 mg kg⁻¹ α-PD-L1. Tumor progression was monitored and recorded using IVIS imaging, and histological analysis was performed to further assess the effects of the treatments on tumor growth. KPC mice, expressing mutant intrapancreatic Kras and Trp53, were obtained from Cyagen Biosciences Inc. (Suzhou, China). Six-week-old female mice were divided into two groups (five mice in each group). One group received oral administration of 100 μL polymyxin B (50 mg kg⁻¹) combined with 0.6 mg kg⁻¹ α-PD-L1 weekly for six weeks. The control group was given 100 μL of PBS with 0.6 mg kg⁻¹ α-PD-L1 on the same schedule. Post-treatment, the mice were sacrificed, and the tumors were resected and weighed.

In vitro effect of LPS

The cytotoxicity of LPS on Panc-02 cells was assessed using an MTT assay. Initially, Panc-02 cells were seeded at a density of 1 × 10⁴ cells per well (100 μL) in a 96-well plate and cultured for 24 hours. Different concentrations of LPS (100 μL) were added to each well, followed by another 24-hour incubation. Subsequently, MTT solution (20 μL, 5 mg mL⁻¹ in PBS) was added to each well, and the cells were incubated for an additional 4 hours. After removal of the culture medium, 150 μL of DMSO was added to dissolve the formazan product. The absorbance at 570 nm was measured using a microplate reader. To evaluate the migration ability of the tumor cells, a cell scratch assay was performed. Panc-02 cells were seeded in a 6-well plate at a density of 5 × 10⁴ cells per well and cultured for 24 hours. Sterile p200 pipette tips were used to create scratches on the plates, and the unattached tumor cells were removed by washing with sterile PBS. Subsequently, 1 mg L⁻¹ of LPS was added to the plates, and the attached cells were co-cultured for another 24 hours. The migration of Panc-02 cells was assessed by capturing photographs using inverted microscopes.

Effect of LPS on in vivo tumor growth and immune response

To investigate the impact of LPS on tumor development and immune response, C57BL/6 mice were transplanted with either 1×10⁶ Panc-02^luc cells with 1 μg g⁻¹ LPS or 1 × 10⁶ Panc-02^luc cells without LPS in their pancreas (five mice in each group). After a period of four weeks, the mice were euthanized, and tumor tissues were collected for histological staining to examine cellular characteristics. Additionally, immune TME analysis was conducted to evaluate the immune response within the tumor tissues.

Screening antibacterial metal ions

To screen for the antibacterial activity of metal ions, four bacterial species (E. coli, S. aureus, B. subtilis, and LGG) were cultured individually for 24 hours. Each bacterial species was then co-cultured with eight different metal ions, each at a concentration of 50 μg mL⁻¹, for 12 hours. After the incubation period, the growth of each bacterial species was measured by recording the OD600.

Antibacterial evaluation of Ga³⁺

To evaluate the antibacterial effectiveness of Ga³⁺ against E. coli, B. subtilis, and LGG, the bacteria were cultured in 10 mL of M9 medium with an initial inoculation dose of 0.1%. Ga(NO₃)₃·6H₂O solutions at a concentration of 50 μg mL⁻¹ were added to the cultures. The OD600 of the cultures was measured after 12, 24, and 36 hours of co-culture to assess bacterial growth inhibition. In addition, E. coli was cultured in M9 medium and treated with Ga(NO₃)₃·6H₂O at a concentration of 50 μg mL⁻¹. Combinations of Ga(NO₃)₃·6H₂O (50 μg mL⁻¹) with TA (50 μg mL⁻¹), EGCG (50 μg mL⁻¹), or GA (50 μg mL⁻¹) were also added to the E. coli culture, respectively. After 24 hours of co-culture, samples from each group were taken and plated onto agar to count bacterial colonies. To determine the uptake of Ga³⁺ by the bacterial cells, the Ga³⁺ concentration was measured using ICP analysis.

Preparation of LGG@Ga-poly

LGG@Ga-poly was prepared using a layer-by-layer self-assembly method. Initially, LGG was cultured in MRS medium for 24 hours and subsequently resuspended in PBS to achieve a concentration of 5×10⁸ CFU mL⁻¹. Next, 10 mL of the LGG suspension was mixed with an equal volume of 5 mg mL⁻¹ chitosan solution and stirred gently for one hour. Following centrifugation at 2239 g for 5 minutes, the bacteria were dispersed in 49 mL of PBS. To prepare the LGG@Ga-EGCG complex, 500 μL each of 40 mg mL⁻¹ EGCG and 10 mg mL⁻¹ Ga(NO₃)₃·6H₂O were rapidly added to the bacterial dispersion. The resulting solution was vigorously stirred for one hour, resulting in the formation of LGG@Ga-EGCG. Subsequently, 10 mL of a 5 mg mL⁻¹ chitosan solution was added to the LGG@Ga-EGCG mixture and stirred for an additional hour to obtain the final product, LGG@Ga-poly.

Environmental tolerance of LGG@Ga-poly

To evaluate the resistance of LGG@Ga-poly in the gastrointestinal (GI) environment, the following experimental procedure was conducted. Initially, a population of 5 × 10⁸ CFU of LGG@Ga-poly was exposed to anaerobic conditions and treated with SGF and SCF at 37 °C for 24 hours. Subsequently, the bacteria were collected and cultured in MRS medium with an initial inoculation of 0.5%. After 24 hours of growth, 150 μL of the culture medium was extracted, and the OD600 was measured as an indicator of bacterial growth.

Ga³⁺ release performance of LGG@Ga-poly

The concentration of Ga³⁺ was determined using ICP analysis, measuring both the initial concentration (C1) and the residual concentration (C2) after the self-assembly process. The loading efficiency (LE) of Ga³⁺ was calculated using the following formula: LE (%) = (C1 - C2) / C1 × 100%. To investigate the release behavior of Ga³⁺ from LGG@Ga-poly in SGF, 1 mL of a 5×10⁸ CFU suspension of LGG@Ga-EGCG or LGG@Ga-poly was placed in dialysis bags with a molecular weight cut-off (MWCO) of 3000. These bags were then immersed in 9 mL of PBS (pH 2.0) and incubated at 37 °C on a shaker. After 4 hours, the dialysates were collected, and the concentration of Ga³⁺ was analyzed using ICP.

Antibacterial activity of LGG@Ga-poly

E. coli, S. aureus, and F. nucleatum were cultured in a 6-well plate for 24 hours. Subsequently, transwell chambers containing 1% LGG@Ga-poly at a concentration of 5 × 10⁸ CFU were placed on the plate. After another 24 hours of co-culture, bacterial growth in the plate was quantified by measuring the OD600. Additionally, 100 μL of bacterial solutions were extracted and spread on agar plates for 24 hours to observe bacterial colonies. To establish a F. nucleatum-infected Panc-02 cell model, Panc-02 cells were seeded in 6-well plates and cultured overnight. Then, the cells were incubated with F. nucleatum at mid-logarithmic phase (6 × 10⁶ CFU) for 1 hour to allow for phagocytosis. After the extracellular bacteria were eliminated by treatment with metronidazole (MTD) (100 μg mL⁻¹) for 2 hours, the bacteria-infected cells were cultured at 37 ^oC for another 24 hours. Then, cells were treated with PBS, Ga(NO₃)₃·6H₂O (50 μg mL⁻¹) or LGG@Ga-poly (5 × 10⁸ CFU) for another 24 hours. Afterwards, cells were washed with sterile PBS and lysed using 0.1% Triton X-100 in a 0.1% BSA aqueous solution for 2 hours. The resulting suspensions were diluted with PBS, and aliquots were plated for analysis.

Antibacterial activity within a bacteria-infected 3D tumor sphere

Panc-01 cells and HPSC cells were cultured as spheroids in a 96-well plate. Specifically, each well was supplemented with 50 μL of 1% agarose solution using microwave heating. Subsequently, Panc-01 cells were added at a density of 4000 cells per well and HPSC cells at a density of 2000 cells per well in 150 μL of agarose gel plate. After one week of growth, well-defined spheroids were observed, which were then replenished with fresh medium to establish 3D PDAC stroma-rich spheroids within another week. To assess the penetration ability, free Cy5 and Cy5-labeled LGG@Ga-poly with equivalent fluorescence intensity were incubated with the spheroids for 24 hours. After removing the medium, the fluorescence of Cy5 was visualized using CLSM at a magnification of 10×. Furthermore, to evaluate the antibacterial efficacy, Mcherry-expressing E. coli (1 × 10⁸ CFU) were co-cultured with the spheroids for 24 hours. Subsequently, either free FITC or FITC-labeled LGG@Ga-poly was added. After an additional 24 hours of incubation, the fluorescence of FITC and Mcherry was visualized using CLSM. Moreover, free Ga³⁺ and LGG@Ga-poly containing an equivalent amount of Ga³⁺ (1 mg mL⁻¹) were incubated with E. coli-infected spheroids for 24 hours. The concentrations of Ga³⁺ and LPS within the spheroids were measured using ICP and an LPS detection kit, respectively.

In vivo biodistribution analysis

Orthotopic Panc-02 tumor-bearing mice were randomly divided into two groups, with three mice in each group. Subsequently, the mice were orally administered 100 μL of either 5× 10⁸ CFU Cy5.5-labeled LGG or Cy5.5-labeled LGG@Ga-poly. The whole-body fluorescence of the mice was monitored at predetermined time intervals using the IVIS system. Upon completion of the experiment, the mice were euthanized, and tumor tissues along with major organs were collected for ex vivo imaging analysis and histological staining. Furthermore, tumors and major organs from the LGG@Ga-poly-treated group were collected and homogenized to measure Ga³⁺ concentration. To evaluate the tumor-penetrating ability, photoacoustic imaging was conducted 12 hours after oral administration of Cy5.5 or Cy5.5-labeled LGG@Ga-poly.

Microbiota analysis

To compare the microbial communities in PDA tumors and healthy pancreases, an orthotopic Panc-02 tumor-bearing mice model was established. The tumors were allowed to progress for one month, after which PDA tumor tissues and a healthy pancreas were collected under sterile conditions. The microbiota present in these samples was analyzed using 16 S rDNA sequencing, a technique commonly used for bacterial community profiling. To assess the impact of LGG@Ga-poly treatment on the intratumor microbiota, tumors were collected after six treatments for microbiota analysis. Additionally, fecal pellets were collected to analyze any changes in the gut microbiota.

In vivo intestinal permeability evaluation

In the in vivo evaluation of intestinal permeability, the potential protective effect of LGG@Ga-poly was investigated through an intestinal permeability assay using FITC-dextran as a marker. After completing the treatment regimen, mice were fasted for 4 hours to establish a consistent baseline condition. Subsequently, the mice were orally administered with 0.6 mg g⁻¹ of 4 kDa FITC-dextran. After an additional 3 hours, peripheral blood samples were collected from the mice, and the fluorescence intensity of FITC in the serum samples was measured to evaluate the intestinal permeability. The fluorescence intensity served as an indicator of the extent of FITC-dextran crossing the intestinal barrier and entering the bloodstream, allowing for the assessment of intestinal permeability.

LPS measurement

Following the administration of different treatments, samples including whole blood, fecal pellets, and tumor tissues were collected and transferred into EP tubes. The blood samples were left to rest at room temperature for 1 hour and then subjected to centrifugation at 861 g for 10 minutes to separate the serum from other components. The obtained serum, along with the supernatants from the fecal and tumoral samples, was collected for measuring the concentration of LPS by a LPS detection kit.

In vivo therapeutic efficacy in a prophylactic setting

The mice were randomly divided into five groups: PBS, LGG, Ga-poly, LGG + Ga-poly, and LGG@Ga-poly (five mice in each group). Prior to tumor cell implantation (Day 0), the mice were administered 100 μL of the respective materials every other day for a total of three treatments (starting from Day −7). Subsequently, the mice received continued treatment every other day for a total of eight treatments. The doses of each material were as follows: LGG (5 × 10⁸ CFU), Ga-poly (5 mg kg⁻¹), LGG + Ga-poly (5 × 10⁸ CFU LGG + 5 mg kg⁻¹ Ga-poly), and LGG@Ga-poly (5 × 10⁸ CFU). On Day 21, the mice were sacrificed, and the tumors were visually inspected and weighed. The T cell infiltration in the tumors was quantified using CD3-immunofluorescence staining. Additionally, six-week-old female KPC mice were divided into two groups (five mice in each group). One group was orally administered LGG@Ga-poly (5 × 10⁸ CFU) every other day, totaling twenty treatments. The control group received 100 μL of PBS on the same schedule. After the treatment period, the mice were sacrificed, and their tumors were resected and weighed.

In vivo antitumor efficacy of LGG@Ga-poly

Mice with Panc-02 tumors were randomly divided into five groups: PBS, LGG, Ga-poly, LGG + Ga-poly, and LGG@Ga-poly (five mice in each group). Starting from Day 0, the mice received various treatments every other day for a total of eleven times, with the same doses as in the prophylactic setting. The tumor volume was monitored using IVIS, MRI, and CT imaging techniques. On Day 28, the mice were sacrificed for ex vivo histological analysis. Furthermore, six-week-old female KPC mice were organized into five groups, each comprising five mice. They underwent various treatments every other day, totaling twenty sessions, with dosages consistent with those used in the prophylactic setting for the Panc-02 model. Following the treatment period, the mice were euthanized, and their tumors were excised and weighed.

Immune TME analysis

The immune cells present in tumor tissues, lymph nodes, and spleen were analyzed quantitatively using flow cytometry after staining. Specifically, the tumor tissues were processed into single-cell suspensions using collagenase IV (Roche), DNAase I (BioFroxx), and hyaluronidase (bioSharp) at 37 °C for 2 hours. The resulting cell suspension was subjected to red blood cell lysis and dispersed in PBS (1 mL). The cell suspension was then filtered through a 70 μm cell strainer and separated using percoll (Stem Cell). The single-cell suspensions were subsequently stained with antibody cocktails, and fluorescently labeled cells were counted using a BD Accuri™ C6 flow cytometer. The immune cell populations that were analyzed included M1-like and M2-like macrophages, MDSCs, DCs, T cells, and memory effector T (T_EM) cells.

Transcriptome analysis

Tumor samples from both the PBS treated group (n = 3) and the LGG@Ga-poly group (n = 3) were excised and promptly preserved at −80^oC before further processing. Total RNA was extracted from the tumor samples by using TRIzol Reagent (Invitrogen), and the genomic DNA was eliminated through DNase I treatment (TaKara). The RNA samples were then assessed for quality using the Agilent 2100 Bioanalyzer. Only high-quality samples, determined by the OD260/280 ratio between 1.8 and 2.2, OD260/230 ratio greater than 2.0, RIN value greater than 6.5, 28 S:18 S ratio greater than 1.0, and RNA quantity exceeding 1 μg, were chosen for constructing the sequencing library. The TruSeq RNA sample preparation kit was employed to build the transcriptome library. Initially, mRNA was selected through the polyA selection method and then fragmented with a fragmentation buffer. The resulting cDNA fragments were then subjected to size selection, where fragments up to 300 bp were selected on a 2% Low Range Ultra agarose gel, followed by PCR amplification with Phusion DNA polymerase (NEB) for 15 cycles. Finally, the sequencing libraries were measured for quantity and quality, then sequenced utilizing the Illumina HiSeq xten/NovaSeq 6000 sequencer with 2 × 150 bp read length. The free online Majorbio Cloud Platform was used to analyze the data of transcriptomic analysis.

In vitro stimulation of RAW264.7 macrophages with LPS

RAW264.7 macrophages were cultured in 6-well plates for 24 hours. Subsequently, the cells were treated with 100 ng mL⁻¹ of LPS and incubated for an additional 24, 48, and 72 hours to evaluate the M2 polarized state using FCM.

Assessment of LGG@Ga-poly combined with LPS in Panc-02 tumor-bearing mice

The mice bearing Panc-02 tumors were divided into two groups: Control and LGG@Ga-poly + LPS (four mice in each group). The mice in the LGG@Ga-poly + LPS group received oral administration of LGG@Ga-poly (5 × 10⁸ CFU) every other day for a total of eleven doses. Simultaneously, LPS (1 μg g⁻¹) was intraperitoneally injected. At the end of the treatment period, the tumors from both groups were collected for FCM analysis and weight measurement.

In vivo antitumor efficacy of LGG@Ga-poly combined with chemotherapy

In the orthotopic Panc-02 tumor-bearing mice, four groups were formed at random: PBS, Gem, LGG@Ga-poly, and LGG@Ga-poly + Gem (five mice in each group). In the PBS group, 100 μL PBS was orally administered to mice every two days for a total of eleven times. In the Gem group, Gem (20 mg kg⁻¹ per mouse) was intravenously administered on the 0^th, 4^th, and 8^th day. In the LGG@Ga-poly group, LGG@Ga-poly (5 × 10⁸ CFU per mouse) was orally administered every two days for a total of eleven times. In the LGG@Ga-poly + Gem group, Gem (20 mg kg⁻¹ per mouse) was intravenously administered on the 0^th, 4^th, and 8^th day, and LGG@Ga-poly (5 × 10⁸ CFU per mouse) was orally administered every two days for a total of eleven times. The mice were sacrificed on Day 28, and tumors were collected for histological analysis.

In vivo antitumor efficacy of LGG@Ga-poly combined with α-PD-L1

The orthotopic Panc-02 tumor-bearing mice were randomly divided into four groups: PBS, α-PD-L1, LGG@Ga-poly, and LGG@Ga-poly + α-PD-L1 (six mice in each group). In the PBS group, 100 μL of PBS was orally administered to mice every two days for a total of eleven times. In the α-PD-L1 group, α-PD-L1 (0.2 mg per mouse) was intravenously injected on the 0^th, 4^th, and 8^th day. In the LGG@Ga-poly group, LGG@Ga-poly (5 × 10⁸ CFU per mouse) was orally administered every two days for a total of eleven times. In the LGG@Ga-poly + α-PD-L1 group, α-PD-L1 (0.2 mg per mouse) was intravenously injected on the 0^th, 4^th, and 8^th day, and LGG@Ga-poly (5 × 10⁸ CFU per mouse) was orally administered every two days for a total of eleven times. The mice were sacrificed on Day 28, and tumors were collected for histological analysis.

In vivo biosafety

The female C57BL/6 mice were randomly assigned into two groups (n = 3). The mice received 100 µL PBS, LGG (5 × 10⁸ CFU per mouse), or LGG@Ga-poly (5 × 10⁸ CFU per mouse) via oral administration, respectively. Three days after treatment, blood samples were collected for liver function tests, including ALT, AST, GGT, TP, ALB, and GLO, kidney function tests, including CRE, TBIL, UREA, and GLU, and hematological tests, including WBC, Lymph, Mid, and Gran.

Statistical analysis

The experimental data were analyzed by GraphPad Prism 8 and the significance level was set at p < 0.05. Each error bar in the figures represents the standard error of the mean (sem) of at least three independent experiments. Two-tailed Student’s t-test was applied to analyze the statistical significance of the difference between two groups, and one-way ANOVA was used for multiple groups. Unless otherwise specified, the mean value was defined as the center value. To minimize bias, the investigators who performed the surgery for cell and in vivo experiments were blinded to the treatment groups.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA sequencing data generated in this study have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under accession code PRJNA1042652. The 16 s rDNA sequencing data generated in this study have been deposited in the NCBI SRA database under accession code PRJNA1041940 and PRJNA1041943. The remaining data are available within the Article, Supplementary Information, or Source Data file. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2019YFA0905603, X.Z.Z.) and the National Natural Science Foundation of China (51988102, 52333004 and 22135005, X.Z.Z.). Figures 1 and 4n, created with BioRender.com, were released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

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Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan, 430072, P. R. China
Zi-Yi Han, Zhuang-Jiong Fu, Yu-Zhang Wang, Cheng Zhang, Qi-Wen Chen, Jia-Xin An & Xian-Zheng Zhang

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Zi-Yi Han
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Zhuang-Jiong Fu
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Yu-Zhang Wang
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Cheng Zhang
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Qi-Wen Chen
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Jia-Xin An
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Contributions

Z.Y.H. and X.Z.Z. conceived the project and designed the experiments. Z.Y.H. and Z.J.F. synthesized materials. Z.Y.H. and Z.J.F. performed in vitro microbiological experiments. Z.Y.H., Y.Z.W., and Z.J.F. performed in vitro cell experiments. Z.Y.H., Y.Z.W., J.X.A., and C.Z. performed in vivo experiments. Z.Y.H., Q.W.C., and Z.J.F. collected and analyzed the data. All authors discussed the results, wrote the manuscript, and reviewed the manuscript.

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Correspondence to Xian-Zheng Zhang.

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Nature Communications thanks Federica Facciotti, Ming-Shiou Jan, Min Zhou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Han, ZY., Fu, ZJ., Wang, YZ. et al. Probiotics functionalized with a gallium-polyphenol network modulate the intratumor microbiota and promote anti-tumor immune responses in pancreatic cancer. Nat Commun 15, 7096 (2024). https://doi.org/10.1038/s41467-024-51534-z

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Received: 10 November 2023
Accepted: 12 August 2024
Published: 17 August 2024
DOI: https://doi.org/10.1038/s41467-024-51534-z

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