Bifidobacterium adolescentis induces Decorin+ macrophages via TLR2 to suppress colorectal carcinogenesis

Background The interplay between gut microbiota and tumor microenvironment (TME) in the pathogenesis of colorectal cancer (CRC) is largely unknown. Here, we elucidated the functional role of B. adolescentis and its possible mechanism on the manipulation of Decorin+ macrophages in colorectal cancer. Methods The relative abundance of B. adolescentis in tumor or para-tumor tissue of CRC patients was analyzed. The role of B. adolescentis was explored in the CRC animal models. The single cell-RNA sequencing (scRNA-seq) was used to investigate the myeloid cells subsets in TME. The expression level of TLR2/YAP axis and its downstream Decorin in macrophages were tested by Western blot and qRT-PCR. Knockdown of Decorin in Raw264.7 was performed to investigate the effect of Decorin+ macrophages on subcutaneous tumor formation. Multi-immunofluorescence assay examined the number of Decorin+ macrophages on the CRC tissue. Results We found that the abundance of B. adolescentis was significantly reduced in tumor tissue of CRC patients. Supplementation with B. adolescentis suppressed AOM/DSS-induced tumorigenesis in mice. ScRNA-seq and animal experiment revealed that B. adolescentis increased Decorin+ macrophages. Mechanically, Decorin was activated by TLR2/YAP axis in macrophages. The abundance of B. adolescentis was correlated with the number of Decorin+ macrophages and the expression level of TLR2 in tumor tissue of CRC patients. Conclusions These results highlight that B. adolescentis induced Decorin+ macrophages and provide a novel therapeutic target for probiotic-based modulation of immune microenvironment in CRC. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-023-02746-6.


Introduction
Colorectal cancer (CRC) ranks third in incidence and mortality globally [1].Gut microbes are closely bound up with CRC via immune cells [2].Accumulating evidence suggested that several special bacteria connected with the initiation and progression of CRC through immune pathway [3][4][5].For instance, Helicobacter hepaticus induces T follicular helper cells and tertiary lymphoid structures to against CRC [6].Among all the immune cells, macrophages are regarded as a critical part interacting with the intestinal flora in CRC.The gut microbiota regulates monocyte-like macrophages accumulation in a chemokine dependent manner and mediates an inflammatory response to facilitate colitis-associated tumorigenesis [7]; Tryptophan metabolites derived from microbes activate the aryl hydrocarbon receptor of tumor-associated macrophages to suppress anti-tumor immunity [8].Accordingly, utilizing the flora to regulate macrophages is a potential therapeutic strategy in CRC [9].
Bifidobacterium plays a critical role in immune maturity and regulation [10][11][12].Studies suggested that Bifidobacterium was associated with enhanced efficacy of checkpoint blockade immunotherapy [13,14].For example, cocktail of Bifidobacterium (B.breve and B.longum) promotes anti-tumor immunity and improves anti-PD-L1 efficacy [15].Intratumoral accumulation of Bifidobacterium cocktail (B.bifidum, B. longum, B. lactis and B.breve) facilitates CD47-based immunotherapy via STING signaling [16].Bifidobacterium pseudolongum enhances the anti-CTLA-4 therapy response by producing the metabolite inosine [17].Most of these studies on cancer focus on Bifidobacterium cocktail and checkpoint blockade immunotherapy.However, the potential role of B.adolescentis on initiation and progression of CRC remains poorly understand.Our previous work indicated that B.adolescentis ameliorated chronic colitis by regulating immune response and gut microbiota remodeling [18].
In this study, we observed that supplementation with B.adolescentis suppressed colorectal tumorigenesis.Mechanically, we uncovered that B. adolescentis induced Decorin + macrophages infiltration in TME and Decorin in macrophages was a crucial molecule mediating the anti-tumor effect of B.adolescentis.In addition, B. adolescentis increased Decorin + macrophages via a TLR2/ YAP-dependent signaling.Our results highlighted the protective role of B.adolescentis on CRC, which provided a new therapeutic strategy based on probiotics modulation of tumor immune microenvironment.

DNA extraction and bacteria DNA quantification
Genomic DNAs extraction and purification from clinical or mice tissue were executed by QIAGEN DNA Mini Kit (Qiagen, Germany) according to the instructions.Quantitative real-time PCR was performed to assess the relative abundance of B. adolescentis.The abundance of B. adolescentis was calculated by -ΔCt method and the universal Eubacteria 16s was used as an internal reference gene.Primers used are listed in the Supplementary Table S1.

Human specimens
A total of 65 paired fresh tumor and adjacent nontumor tissue were obtained from patients with primary CRC who had not received preoperative anti-tumor and antibiotic treatment from Sir Run Run Shaw Hospital of Zhejiang University (Hangzhou, China).All samples were frozen in liquid nitrogen until use.Clinical Research Ethics Committee of the Sir Run Shaw Hospital, Zhejiang University School of Medicine approved the protocol.All aspects of the study were conducted in accordance with the principles of the Declaration of Helsinki.

Carcinogen-induced cancer model
6-week-old male C57BL/6 wildtype mice were obtained from Shanghai SLAC Laboratory Animal, China.Before bacterial intragastric administration, as previous study reported, 2 mg/mL streptomycin (Cat.#MB1275, Meilunbio) solubilized in water was given to mice for 7 days to ensure the conformance of regular microbiota and facilitate B. adolescentis colonization.C57BL/6 mice were given one single intraperitoneal injection of carcinogen azoxymethane (AOM, Sigma) (10 mg/ kg per mouse), and then were given 5 successive days of 2.5% dextran sodium sulfate (DSS, MP Biomedicals) in the drinking water, following by regular drinking water for 2 weeks.This cycle was then repeated for twice [19].Two weeks later, mice in B.adolescentis and E. coli groups were administrated with 1 × 10 9 CFU B.adolescentis or E. coli suspended in 200 μL sterile PBS every day, and the control group was administered with PBS.After about 3 months, mice were sacrificed and the colorectum was surgically excised for further analysis.

Animal use and care
All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Zhejiang University (ZJU).All animal experiments strictly adhered to protocols, policies, and ethical guidelines formulated by our IACUC.BALB/c nude mice, BALB/c mice and C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal, China.All mice were maintained in ventilated cages with 12 h light/dark cycles, constant temperature and humidity, enriched water and ad libitum feeding under specific pathogen-free (SPF) conditions.

Isolation of primary macrophages
For primary mouse macrophages BMDMs, bone marrow cells were harvested from 8-10 weeks old C57BL/6 mice as previously done [20].6 days later, BMDMs were confirmed by flow cytometry for CD11b and F4/80 and further used.For primary human macrophages, after isolating the CD14 + monocytes from PBMC using the MojoSort Human CD14 Selection Kit (Biolegend Cat No:480025) according to the manufacturer's protocol, we cultured them in RPMI 1640 supplemented with 10% (v/v) FBS, penicillin/streptomycin, and 50 ng/mL M-CSF for 5 days to induce macrophages differentiation [21].For polarization of M1 macrophages, BMDMs and THP-1 cells were treated by 100 ng/mL IFN-γ (Cat.#C746, Novoprotein) for 24 h as previous study reported [22], then the expression of aimed genes were examined by qRT-PCR.

Cytotoxicity experiments
For cytotoxicity experiments, BMDMs were pretreated with B.adolescentis, E. coli or vehicle (PBS) for 24 h, and then co-cultured with HCT116 or CT26 (BMDMs: tumor cells = 1:1) for 24 h.The level of LDH was evaluated according to the manufacturer's instructions [23].

Isolation of colorectal lamina propria cells
Isolation of colorectal lamina propria cells was performed as previously done [20].In brief, the posterior 1/3 of the colorectum without lymph and adipose tissue was cut into small pieces and washed in RPMI 1640 medium (GIBCO, China).The tissue was incubated in D-Hanks (Cat.#MA0039, Meilunbio) buffer supplemented with 1 mmol/L DTT (Cat.#MB30471, Meilunbio) and 5 mmol/L EDTA (Cat.#MB2514, Meilunbio) on a shaker (200 rpm) for 30 min at 37℃.And then the remaining colorectum was cut into 1 mm pieces and further digested in Hanks buffer (Cat.#MA0041, Meilunbio) supplemented with 1 mg/mL Type IV collagenase (Cat.#A005318, Sangon Biotech) for 30 min at 37℃ with 200 rpm shaking.After complete digestion, the cell suspension was passed through a 300-mesh filter and then centrifuged at 500 g for 5 min.The isolated colorectal lamina propria cells were further analyzed.

Cell culture in presence of B.adolescentis
BMDMs, THP-1 cells or Raw264.7 cells were seeded in 6-well plate at a density of 5 × 10 5 cells per well and cultured in DMEM or RPMI 1640 medium with 10% (vol/vol) FBS overnight.BMDMs, THP-1 cells or Raw264.7 cells were co-cultured with B.adolescentis at a MOI = 100:1 for 24 h.Finally, the protein and RNA of BMDMs, THP-1 cells or Raw264.7 cells were extracted for analysis.

RNA extraction and quantitative qRT-PCR
RNAs were extracted from macrophages or tissue samples using Trizol reagent (Invitrogen, USA), and total RNA were reversed by Evo M-MLV RT Kit (Accurate Biology, China) according to the manufacturer's instructions.Quantitative RT-PCR analysis was performed in triplicate in ROCHE LightCycler480 System (Rotor gene 6000 Software, Sydney, Australia) with SYBR Premix Ex Taq (Takara, Japan).Relative abundance was demonstrated by -ΔCt method.Primers used are listed in the Supplementary Table S1.

Genomics library and single cell RNA sequencing
Primary Sequencing data were demultiplexed and converted to FASTQ format by Illumina bcl2fastq software.Cell Ranger pipeline (version 3.1.0)was used to perform sample demultiplexing, barcode processing and single-cell 3, gene counting.Then scRNA-seq data were aligned to Ensemble genome GRCm38 reference genome.After filtering and UMI (universal molecular identifier) counting, a total of 18,661 single cell captured from 2 samples were captured using 10 × Genomics Chromium Single Cell 3' Solution.The mean reads per cell was 49,151 with median UMI Counts per Cell of 5,790 after aggregation.
To further identify sub-clusters within myeloid cell type, we re-analyzed cell type annotated as myeloid cells.Specifically, subset of Seurat object was extracted from the original expression matrix by default Seurat function, then we re-performed dimensionality reduction and PCA analysis and generate clusters as described above.Sub-cell types identified from sub-clustering of the myeloid cells were performed by "SingleR" package with "ImmGen" as the reference database, which identified Neutrophils, Macrophages, Monocytes, DC, Basophils cells.Finally, differentially expressed genes between PBS and B.a group in each sub-cell subsets were determined by Seurat package and visualized by "pheatmap" R packages.

RNA sequencing
For RNA sequencing, BMDMs were co-cultured with B.adolescentis or vehicle (PBS) (MOI of 100:1) for 24 h.Total RNA was extracted from cells by Trizol reagent (Invitrogen, USA), and mRNA was purified by poly-T oligo-attached magnetic beads.Sequencing libraries were generated using NEBNext ® Ultra ™ RNA Library Prep Kit for Illumina ® (NEB, USA) following manufacturer's instruction.150 bp paired-end libraries were sequenced by Illumina PE150 platform.Paired-end reads were aligned to the human genome version hg19 using Hisat2 v2.0.5.Differential expression analysis of two groups was performed using the DESeq2.P value < 0.05 and | log2 (fold change) |> 0.5 were considered as significant.

Histopathologic analysis
Colorectal tumors were fixed with 4% paraformaldehyde overnight at room temperature and embedded in paraffin.Hematoxylin and eosin (H&E) stain was performed as previously described.

Statistical analysis
Statistical analysis was performed using the GraphPad Prism 9.0 software.Data were analyzed with paired or unpaired Student t test, Mann-Whitney test, ANOVA test or linear regression as showed in figure legends.P value less than 0.05 was considered statistically significant.

Supplementation of B. adolescentis suppressed colorectal tumorigenesis in mice
We observed that the abundance of B.adolescentis was significantly lower in tumor tissue from patients with CRC than in para-tumor (Supplementary Fig. 1A).To investigate the role of B.adolescentis in CRC, we utilized the AOM/DSS-induced CRC model in C57BL/6 mice, which were pretreated with streptomycin (2 mg/mL) for 7 days and administrated with B.adolescentis, Escherichia coli (E.coli) or vehicle (PBS) control (Supplementary Fig. 1B,  C), and found that supplementation with B.adolescentis resulted in considerably fewer and smaller tumors than E. coli or vehicle group (Fig. 1A-C).The expression of cell proliferation marker Ki67 and tumor angiogenesis marker CD31 were lower in colorectal tumor tissue from B.adolescentis-treated mice (Fig. 1D-F).Collectively, these data indicated that B.adolescentis could suppress colorectal tumorigenesis in vivo.

B.adolescentis recruited macrophages to suppress colorectal tumorigenesis
To understand the potential mechanism by which B.adolescentis suppresses CRC, we profiled the tumor from above AOM/DSS model using single-cell RNA sequencing (scRNA-seq) (Fig. 2A) and focused on the myeloid-derived immune cells (Supplementary Fig. 1D), as previous studies have shown that intratumoral Bifidobacterium facilitates innate immune response and scRNA-seq analyses informed the key role of myeloidtargeted therapies in CRC [16,24].We then divided myeloid-derived immune cells into five groups based on their top expression markers (Fig. 2B, Supplementary Fig. 1E) and found that B.adolescentis group exhibited an increase in the proportion of intratumoral macrophages and basophiles, and a decrease in dendritic cells (DCs) and monocytes (Fig. 2C, Supplementary Fig. 1F).
An increased percentage of macrophages was observed in colorectum lamina propria (CLP) and blood of AOM/DSS model treated with B.adolescentis when compared to E.coli or vehicle groups by flow cytometry assays (Fig. 2D, E, Supplementary Fig. 3A), whereas the percentage of DCs had no significant difference (Supplementary Fig. 3B).Similar results were confirmed in mice tumor tissue by immunofluorescence staining (Fig. 2F, G).Meanwhile, we co-cultured BMDMs or THP-1 cells with B.adolescentis for 24 h in vitro and found that B.adolescentis-treated macrophages exhibited a dramatic cell migration compared to E.coli or vehicle groups (Supplementary Fig. 3C, D).
To further determine the effect of B.adolescentistreated macrophages on CRC progression, we injected HCT116 combined with B.adolescentis, E. coli or vehicle treated THP-1 cells (HCT116: THP-1 = 10:1) into BALB/c nude mice, and found that B.adolescentis-treated macrophages suppressed the tumor growth compared with E. coli or vehicle groups (Fig. 2H-J).In consideration of the extensive crosstalk among immune cells, the subcutaneous tumor experiment was performed in BALB/c immune-competent mice using CT26 and Raw264.7.B.adolescentis-treated macrophages consistently suppressed the tumor growth compared with E. coli or vehicle groups (Supplementary Fig. 3E-G).These data suggested that B.adolescentis promoted macrophages infiltration and had a suppressive effect on CRC.

B.adolescentis facilitated the infiltration of Decorin + macrophages to suppress CRC
For exploration of the underlying mechanism by which B.adolescentis-treated macrophages exert anti-tumor function, we performed RNA-seq in BMDMs with or without B.adolescentis treatment (Fig. 3A), and its differentially expressed genes were analyzed and then overlapped with the differentially expressed genes of macrophages in vivo from the scRNA-seq mentioned above, and finally obtained 11 genes (Fig. 3B, C, Supplement Fig. 4A).We verified that Decorin (Dcn) had the highest fold change of expression in BMDMs treated with B.adolescentis in vitro (Fig. 3D).Furthermore, immunofluorescence staining showed that B.adolescentis increased the DCN + macrophages (F4/80 + DCN + ) in tumor tissue of AOM/DSS model (Fig. 3E, F).Meanwhile, we analyzed the expression of Dcn in different cells subtypes in scRNA-seq, and found that macrophages exhibited higher Dcn expression compared to other cell subsets (Supplementary Fig. 4B).
We found a significant increase in Dcn + macrophages in B.adolescentis group through scRNA-seq data.(Supplementary Fig. 4C).Western blot analysis also confirmed that B.adolescentis increased the expression level of DCN in macrophages (Fig. 3G).Studies have reported that Dcn as a multivalent therapeutic agent against cancer by engaging multiple receptor tyrosine kinases like EGFR, Met and VEGFR2 [25], and Dcn deficiency promoted epithelialmesenchymal transition and colorectum cancer metastasis  E, G, I, J) (See figure on next page.)[26].To determine whether the tumor-suppressive effect of B.adolescentis on CRC relies on DCN + macrophages, we established the Dcn-KD Raw264.7 cells by lentivirus-based Cas9 system (Fig. 3H, I).We then injected CT26 cells combined with Dcn-KD or wildtype Raw264.7 cells (CT26: Raw264.7 = 10:1) treated with or without B.adolescentis (MOI = 10:1) into BALB/c nude mice, and found that B.adolescentis-treated Raw264.7 obviously impeded tumor growth, whereas knockdown of Dcn notably diminished the tumor suppressive effect of B.adolescentis on CRC (Fig. 3J-L).We found B.adolescentis-treated macrophages had stronger cytotoxic activity against tumor cells than E. coli or vehicle (PBS) groups in vitro (Supplementary Fig. 4D, E).The levels of Il6 and Tnf were elevated in B.adolescentis-treated macrophages, while Dcn deficiency did not influence the expression of Il6 and Tnf (Supplementary Fig. 4F, G).Taken together, these data suggested that B.adolescentis induced DCN + macrophages to suppress CRC growth.

The activation of TLR2 is essential for inducing DCN + macrophages by B.adolescentis
Pattern recognition receptors (PRRs) play a central role in innate immune response [27,28].As Toll-like receptors (TLRs) are a major class of PPRs that recognize extracellular signals, we screened out TLRs that were differentially expressed in RNA-seq of B.adolescentistreated BMDMs (Fig. 4A) and checked them by qRT-PCR, and discovered that TLR2 was most significantly upregulated (Fig. 4B).We then confirmed the upregulation of TLR2 in BMDMs treated with B.adolescentis at the protein level (Fig. 4C).Mechanically, to verify that B.adolescentis induced the DCN + macrophages via TLR2, we performed TLR2 inhibition assays by Cu-CPT22 and found that Cu-CPT22 administration abolished the upregulation of DCN in BMDMs treated with B.adolescentis (Fig. 4D, E).Similar results were reproduced in THP-1 cells (Fig. 4F-I).We further combined injected HCT116 and THP-1 cells with B.adolescentis (MOI = 10:1) or vehicle into BALB/c nude mice along with intraperitoneally injecting Cu-CPT22 every two days.We found that TLR2 inhibition reduced the number of DCN + macrophages in tumor and attenuated tumor suppressive effect of B.adolescentis-treated macrophages (Fig. 4J-M, Supplementary Fig. 5A-C).These results suggested that B.adolescentis induced the DCN + macrophages in a TLR2-dependent way.

B.adolescentis regulated DCN + macrophages through TLR2/YAP axis
Previous studies have demonstrated that TLR2/YAP axis regulated cancer innate immunity [29,30], yet the function of TLR2/YAP axis in macrophages was unclear.
We discovered that the level of YAP was upregulated in BMDMs and THP-1 cells (Fig. 5A, B) and TLR2 inhibition diminished the regulation of B.adolescentis on YAP (Supplementary Fig. 6A).The nuclear expression of YAP increased in BMDMs and THP-1 cells after B.adolescentis treatment (Fig. 5C-E).YAP inhibitor verteporfin markedly reduced the expression of YAP and DCN in B.adolescentis-treated macrophages but it did not influence the expression of TLR2 (Fig. 5F, G, Supplementary Fig. 6B, C).And overexpression of YAP in BMDMs and THP-1 cells increased the level of DCN (Fig. 5H, I).We then injected HCT116 combined with B.adolescentis (MOI = 10:1) or vehicle treated THP-1 cells into BALB/c nude mice, and intraperitoneally injected with verteporfin every days.YAP inhibitor administration attenuated the tumor suppressive effect of B.adolescentis-treated THP-1 cells and reduced the number of DCN + macrophages in tumors on BALB/c nude mice.(Fig. 5J-L, Supplementary Fig. 6D-F).Furthermore, we found that the expression levels of DCN, TLR2 and YAP were elevated after treated with B.adolescentis in human primary macrophages (Supplementary Fig. 7A, B).
To investigate the relationship between DCN + macrophages and macrophage polarization state, we examined the related markers of macrophages after co-culturing with B.adolescentis and found that the number of M1 macrophages increased while M2 macrophages decreased in B.adolescentis-treated BMDMs (Supplementary Fig. 8A-D).B.adolescentis upregulated the expression levels of DCN, TLR2 and YAP in M1 macrophages (Supplementary Fig. 8E, F), which reflected that the function of DCN + macrophages might be similar with traditional M1-like macrophages.These results reflected that B.adolescentis increased DCN + macrophages through the TLR2/YAP axis.

B.adolescentis abundance correlated with TLR2 and DCN + macrophages in patients with CRC
To further investigate the clinical relevance, we quantified B.adolescentis and the expression of TLR2 and DCN with quantitative qRT-PCR analysis in paired fresh CRC and adjacent non-tumor tissue.According to the level of B.adolescentis in CRC tissue, we divided these patients (n = 65) into high and low B.adolescentis group.We found that compared with the low B.adolescentis group, the number of macrophages in high B.adolescentis group was higher (Fig. 6A), and the abundance of B.adolescentis was related to the number of DCN + macrophages (Fig. 6B).Moreover, the expression of DCN and TLR2 was decreased in CRC tissue compared with their adjacent non-tumor tissue (Fig. 6C), and the abundance of B.adolescentis was positively correlated with the expression of TLR2 and DCN in CRC tissue (Fig. 6D).In addition, the expression of DCN was correlated with the expression of TLR2 (Fig. 6E).Lastly, we confirmed the above results using TCGA GTEx datasets (Fig. 6F, G).These data indicated that B.adolescentis abundance correlated with the expression of TLR2 and the number of DCN + macrophages in patients with CRC.

Discussion
Several infant-type species of human-derived bifidobacterial (HRB) are associated with strengthening host immunity and suppressing tumorigenesis.However, the role and mechanism of adult-type HRB in cancer remains unclear [31].In this study, we revealed that B.adolescentis suppressed colorectal tumorigenesis in AOM/DSS mice.
Macrophages are a major component of TME whose functional plasticity leads to anti-tumor and pro-tumor function in different environment [32].Furthermore, targeting macrophages is an emerging field of interest due to synergies with immune checkpoint inhibitors, chemotherapy, and radiation therapy in preclinical studies [33,34].Our groups have reported that some specific microbes were closely related to macrophages functions [20,35].In this study, we discovered that B.adolescentis suppressed colorectal tumorigenesis through inducing DCN + macrophages, and targeting macrophages with B.adolescentis maybe a potential novel strategy for cancer therapy.
Decorin is a member of the small leucine-rich proteoglycans (SLRPs) which are an important subset of extracellular matrix [36].Soluble DCN engages multiple receptor tyrosine kinases within the target-rich environment of the tumor stroma and tumor parenchyma [25].DCN possesses a multitude of oncosuppressive functions including growth suppressing, angiostasis, and tumor cells mitophagy arrest [37].Several research groups have discovered that DCN deficiency promotes epithelial-mesenchymal transition and hepatic metastasis of colorectal carcinoma [26,37].DCN derived from T cells mediates inhibition of carcinogenesis in microglia and reduces glioma formation [38].However, the function of macrophage-derived DCN has not been reported.We found that B.adolescentis increased the DCN + macrophages and then suppressed colorectal tumorigenesis.B.adolescentis also boosted the cytotoxicity of macrophages against tumor cells and increased the expression of Il6 and Tnf in macrophages in a Dcn-independent way.
Toll-like receptors (TLRs) are an important component family of pattern recognition receptors (PRRs) responsible for early recognition in the innate immune system.TLR2 recognizes a vast of ligands derived from microbes such as lipoproteins/lipopeptides and peptidoglycans [39].Bifidobacterium breve has been reported to induce dendritic cell maturation, activation, and survival via TLR2 [40].Here, we found that B.adolescentis elevated the expression of TLR2 in macrophages and the activation of TLR2 is essential for B.adolescentis regulating DCN + macrophages.However, how B.adolescentis was recognized by TLR2 in macrophages needs to be further explored.
Emerging evidences suggested that the differentiation, metabolism, and functions of innate immune cells were extensively regulated by YAP [41,42].Microbes are reported to regulate the activation of YAP in cancer cells to inhibits colorectal tumorigenesis [43,44].YAP in macrophages promoted the LPS/IFN-γ-triggered activation of M1 macrophages, while impaired the IL-4/IL-13 induced M2 macrophages polarization [45].Prior studies suggested that TLR2/YAP axis played an important role in innate immunity and the activation of TLR2/YAP axis in tumor cells suppressed tumor growth [29,30].However, the underlying mechanism of B.adolescentis interacted with macrophages and the function of TLR2/YAP axis in macrophages remain largely unknown.Indeed, we observed that TLR2/YAP axis was activated by B.adolescentis and inhibition of TLR2/YAP axis significantly impaired the induction of DCN + macrophages by B.adolescentis.Consistent with our findings, B.adolescentis, TLR2 and DCN were reduced in CRC tissue.However, the relationship between the expression of TLR2 and DCN in macrophages and the abundance of B.adolescentis needs to be further explored.
In conclusion, our results provided a novel insight into the interaction and molecular mechanism of Bifidobacterium with macrophages.Our findings suggested that B.adolescentis could act as probiotics to regulate macrophages in colorectal cancer, thus becoming a potential target for tumor vaccine delivery and providing a novel strategy for tumor therapy.

(Fig. 4 Fig. 5 B
Fig. 4 The activation of TLR2 is essential for inducing DCN + macrophages by B.adolescentis.A The heatmap of differentially expressed TLRs genes in RNA-seq of BMDMs treated with B.adolescentis or vehicle (PBS).B The levels of differentially expressed TLRs genes in BMDMs treated with B. adolescentis were determined by qRT-PCR.C BMDMs were incubated with B.adolescentis or vehicle (PBS) for 24 h.Protein levels of TLR2 and DCN were tested by Western blot.D, E BMDMs were incubated with B.adolescentis or vehicle (PBS) for 24 h with or without 25 μM Cu-CPT22.Protein levels of TLR2 and DCN were tested by Western blot and mRNA level of Dcn was tested by qRT-PCR.F-I THP-1 cells were incubated with B. adolescentis or vehicle (PBS) for 24 h (H-G).THP-1 cells were incubated with B.adolescentis or vehicle (PBS) for 24 h with or without 25 μM Cu-CPT22 (H, I).Protein levels of TLR2 and DCN were tested by Western blot and mRNA level of DCN was tested by qRT-PCR.J-L HCT116 cells were injected into BALB/c nude mice combined with THP-1 cells pretreated with B.adolescentis or vehicle (PBS) for 24 h (n = 5 per group).From the beginning of tumor inoculation until sacrifice, 3 mg/kg Cu-CPT22 or vehicle (5% DMSO) were injected intraperitoneally to mice every two days.Tumor volume was recorded after 6 days.M The positive ratio of Ki67 in mice tumor tissue.The independent experiment was repeated three times.Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; Student t test (B, G), ANOVA test (E, I, K, L, M)

Fig. 6 B
Fig. 6 B.adolescentis abundance correlated with TLR2 and DCN + macrophages in patients with CRC.A Representative images of immunofluorescence assay for CD68 in tumor tissue with low or high B.adolescentis abundance from cohort; scale bars, 50 μm.B Representative images of immunofluorescence staining of DCN + macrophages (CD68 + DCN + dual positive) in tumor tissue of CRC; scale bars, 50 μm.The yellow and white arrows indicate the positively stained cells.C qRT-PCR analysis of DCN and TLR2 mRNA expression in tumor or para-tumor tissue of CRC.D The correlation of B.adolescentis with DCN or TLR2 was analyzed in tumor tissue of CRC.E The correlation of DCN and TLR2 was analyzed in tumor tissue of CRC.F Transcript levels of DCN and TLR2 in colorectal cancer and normal tissue from TCGA database.G The correlation of DCN and TLR2 was analyzed in TCGA database.The independent experiment was repeated three times.Data are shown as mean ± SD. * P < 0.05, **** P < 0.0001; Mann-Whitney test (A, C, F), Linear Regression (D, E, G) (See figure on next page.)