Human sample collection
Fresh stool samples were obtained from 71 patients with CRC and 40 healthy subjects at the Sir Run Run Shaw Hospital of Zhejiang University School of Medicine (cohort 1). Fresh tumor and paired normal tissues were obtained from 99 patients with CRC who underwent surgical resection at Sir Run Run Shaw Hospital (cohort 2). All samples were refrigerated at liquid nitrogen until use. Fresh CRC tissues and their paired normal tissue were obtained from 20 patients with CRC who underwent surgical resection at Sir Run Run Shaw Hospital (cohort 3). The tissue array tissues came from the patients with CRC at the Second Affiliated Hospital of Zhejiang University School of Medicine (cohort 4). Clinical Research Ethics Committee of the Sir Run Shaw Hospital, Zhejiang University School of Medicine approved the protocol (20211103-35). All aspects of the study were conducted in accordance with the principles of the Declaration of Helsinki.
GMrepo database analysis
The GMrepo revealed the relative abundance of human gut microbiota in the people with different diseases. The abundance data of B. a in the patients with CRC and the healthy people were downloaded from GMrepo database using GMrepo RESTful APIs for R (version 3.6.1 https://www.r-project.org) and the RStudio (version 1.1.442 https://www.rstudio.com) software. We assessed data quality by consulting the description of the samples and supplementary data of related publications. Then, the relative abundance of B. a for the healthy and CRC patients was analyzed.
Bacteria strain and culture
B. adolescentis ATCC15703 was purchased from American type culture collection (ATCC, USA). V4 of 16S ribosomal RNA sequencing was performed to confirm bacterial strain at the species level. The bacteria were cultured in anaerobic modified Reinforced Clostridium Medium (BD Difco, Sparks, MD, USA) under an atmosphere of 10% H2, 10% CO2, and 80% N2 (AW500SG anaerobic workstations, ELECTROTEK, England) for 48 h. The non-pathogenic commensal intestinal bacteria, E. coli strain DH5a (Code No.9057, Takara), which was used as a negative control, was cultured in Luria-Bertani medium (Cat. #A507002 Sangon Biotech) at 37℃. When the optical density (OD) at 600 nm of B. a reached 1.0, the cultures were centrifuged at 3000 rpm for 5 min at 4℃ and then washed twice with sterile anaerobic PBS, then resuspended at a final concentration of 1×109 CFU/300µl under strictly anaerobic conditions.
Animal use and care
All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Zhejiang University (ZJU) (IACUC-02102214). All animal experiments strictly adhered to protocols, policies, and ethical guidelines formulated by our IACUC. ApcMin/+ mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (NBRI), China. BALB/C nude mice and C57L/B6 mice were purchased from Shanghai SLAC Laboratory Animal, China. All mice were maintained in ventilated cages with 12-hour light/dark cycles, constant temperature and humidity, enriched water and ad libitum feeding under specific pathogen-free (SPF) conditions.
Carcinogen-induced cancer model
6-week-old male C57BL/6 wildtype mice were purchased from Shanghai SLAC Laboratory Animal, China. Before bacterial intragastric administration, mice were fed with 2 mg/ml streptomycin (Cat. #MB1275, Meilunbio) in the drinking water for 7 days to ensure the consistency of regular microbiota and facilitate B. a colonization as previous study reported. Mice were given a cycle of one single intraperitoneal injection of azoxymethane (AOM, Cat. #A5486, sigma, 10 mg/kg body weight) at first week, then followed by three cycles of 5 days of 2.5% DSS (Cat. #160110, MP Biomedicals) administration. At the intervals, mice were performed administration of 1×109 colony forming units (CFU) of B. a, E. coli or the same volume of PBS three times per week for 120 days for the development of neoplastic lesions.
Spontaneous adenomatous mice
Prior to intragastric bacteria administration, Male C57BL/6J ApcMin/+ mice (6-8 week of age, 20g) were fed with 2 mg/mL streptomycin in the drinking water for 7 days to ensure the consistency of regular microbiota and facilitate B. a colonization. ApcMin/+ mice were randomly assigned to three groups. Mice in B.a and E. coli groups were administrated 1×109 CFU B. a or DH5a suspended in 300µl sterile anaerobic PBS, respectively, every 2 days for 3 months. The control group was administrated with PBS. Two cycles of 10-day 1% DSS was given to accelerate tumorigenesis. The body weight of mice was measured every week and mice anus prolapse were observed at the final month.
At the indicated time intervals, colon and spleen tissues were harvested after fasting. Colon tissues were photographed and the number and size of tumors were measured. Tumor sizes (diameter) were quantified as <1 mm, 1-2 mm, 2-3 mm, or >3 mm. Tumor load was calculated as the sum of all tumor diameters in a single mouse. Spleen tissues were photographed and weight measured.
Genomics library and single cell RNA sequencing
The prepared single-cell suspensions were loaded to 10× Chromium to capture enough single cell according to the 10×Genomics Chromium Single-Cell 3’ kit (V3) manufacturer’s instructions. The reverse transcription, cDNA amplification and library construction steps were performed according to the standard protocol (10× Genomics). Sequencing libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run,150bp) by LC-Bio Technology co.ltd (HangZhou, China) at a minimum depth of 20,000 reads per cell.
We excluded contaminating lineage cells and low-quality cells, and finally obtained 14,736 cells. Unsupervised graph clustering divided infiltrating cells into 24 groups based on gene expression pattern. Cells were divided into 6 groups according to their top expression markers, as previous studies reported[24-26]. T cells were marked by Cd3e, Cd3d, Cd3g, Trbc1, Trbc2, Icos. Myeloid cells were marked by Cd14, Csf1r, Fogr3, Adgre1. Fibroblast cells were Col1a2, Col1a1, Dcn, Sparc. Cancer cells were Epcam, Cdh1, Krt8, Krt18. Endothelial cells were marked by Pecam1, Cdh5, Ramp2, Eng. B cells were marked by Cd79a, Cd79b, Cd19, Ms4a1. According to the Fibroblast cell group, unsupervised graph clustering divided infiltrating cells into 12 groups based on gene expression. Cell types ﬁnally identiﬁed were plotted in t-SNE.
DNA extraction and bacteria DNA quantification
Bacterial DNA from human fecal contents were extracted using QIAGEN stool kits (Cat. #51604, QIAGEN, Germany), and bacterial genomic DNA from human tissues were extracted using QIAGEN DNA mini kits (Cat. #56304) according to the manufacturers’ protocol. Quantitative real-time PCR was performed to assess the B. a genes, Universal Eubacteria 16S and PGT using a ROCHE LightCycler®480 System (Rotor gene 6000 Software, Sydney, Australia). Each reaction was performed in triplicate with SYBR Premix Ex Taq (Cat. #RR820A, Takara, Japan), primers and 100ng template gDNA. Relative abundance was calculated by -ΔCt method. Universal Eubacteria 16s was used as internal reference gene for stool samples. The PGT gene was used as internal control for tissue samples. Primers used are listed in the Supplementary Table S1.
Subcutaneous tumor models
Female BALB/C nude mice (3-4 week of age, 15 g) were kept in SPF conditions. CAFs from CRC patient were incubated with B. a (MOI=10:1) for 48 hours, then HCT-116 cells and CAFs were washed twice with PBS and harvested using trypsin-EDTA solution (Cat. # GNM25200, Genom). 3×106 HCT-116 cells were mixed with B.a treated-CAFs (MOI=10:1) or PBS treated-CAFs with 25 µL matrigel matrix (Cat. #354234, Corning Biocoat), and then injected (100 µL per mouse) subcutaneously into the right flank of nude mice. After 7 days implantation, tumor volume was monitored every two days and calculated as follows: Volume=0.54×L×W2, where L is the longest diameter and W is the shortest diameter. At the terminal time, the tumor weights were recorded.
Colorectal tumors or subcutaneous tumors were fixed overnight with 10% formalin at room temperature and then embedded in paraffin. Sections of 5 μm were stained with hematoxylin and eosin (H&E) for pathological analysis. For immunohistochemistry, sections of paraffin-embedded tissue were stained by PCNA-specific antibody (Cat. #GB11010-1, diluted 1:1000, Servicebio, China), α-SMA-specific antibody (Cat. #GB13044, diluted 1:1000, Servicebio), CD31-specific antibody (Cat. #GB11063-2, diluted 1:1000, Servicebio) and Ki67-specific antibody (Cat. #GB111141, diluted 1:1000, Servicebio), and visualized by DAB (Cat. #G1212, Servicebio) staining according to manufacturer’s instructions.
Tumor sample dissociation and single cell suspension preparation
Mice colon tumor tissues were acquired from AOM/DSS model with or without B. a. Tumors were cut into 0.5 mm2 fragments. Tumor samples were digested for 40 min at 37 °C in Hanks buffer (Cat. #MA0041, Meilunbio) containing 5% FBS (Cat. #10270, Gibco), Collagenase IV (200U/ml, Cat. #A005318, Sangon Biotech) and DNase I (120 U/ml, Cat. #B300065, Sangon Biotech), as previous study reported. Samples were typically fully dissociated at this step and filtered through a 40 mM cell strainer. Cells were centrifuged down at 500 g for 10 min and resuspended in 1 mL of ACK lysis (Cat. #R1010, Solarbio) and placed on ice for 3 min. Cells were spun down at 700 g for 5 min and placed on ice. The dead cells were removed by Dead Cell Removal MicroBeads (Cat. #MACS 130-090-101, Germany) and Miltenyi ® Dead Cell Removal Kit (Cat. #MACS 130-090-101, Germany). In the end, the cells were resuspended in PBS containing 0.04% BSA and centrifuged at 300 g for 3 min at 4 °C. The cell viability needed to be above 85% determined by trypan blue staining (Cat. #MA0130, Meilunbio) using an automated counter, finally subsequently adjusted the cell concentration to 700-1200 cells/µL.
HCT-116 human colon cancer cells were obtained from the ATCC at the beginning of this project. Cells were maintained at 37 ℃ under 5% CO2 in McCoy’s 5A (Cat. #GNM16600, Genom) with 10% (vol/vol) FBS (Cat. #10270, Gibco) supplemented with 1% (vol/ vol) penicillin and streptomycin (Cat. #P1400, Solarbio) and maintained in culture for a maximum of 2 months or 10 passages. Murine embryonic fibroblast NIH/3T3 were also obtained from the ATCC at the beginning of our study and maintained in culture for a maximum of 2 months or 10 passages. All cells tested negative for Mycoplasma contamination and were authenticated on the basis of short tandem repeats fingerprinting before use.
Fibroblast (CAFs and normal tissue–associated fibroblast) isolation
The fresh tumor or normal colon tissues were cut into small blocks with a diameter of 2 mm and subsequently seeded on the surface of culture flask containing DMEM supplemented with 20% FBS (Gibco) and 1% penicillin/streptomycin (Solarbio). Culture flask was inverted and maintained at 37 ℃ under 5% CO2 for 1 hour until the culture flask was turned over. The adherent cells were continuously cultured in DMEM with 20% FBS for approximately 2 weeks (tumor tissues) or 3 to 4 weeks (paired normal colon tissues). The culture medium was replaced every three days. Large groups of fibroblasts (morphologically spindle-shaped cells became apparent after 2 weeks and they were validated by western blot and immunofluorescence staining.
Cell culture in the presence of B. adolescentis
CAF cells were digested by trypsin-EDTA solution (Cat. #GNM25200, Genom) slightly, then they were seeded at a density of 2×105 cells per well in 6-well plate and cultured in DMEM with 10% (vol/vol) FBS (Gibco) overnight. Then they were incubated with B. a at a MOI of 100:1 for 48 h. Finally, CAF cells were digested by trypsin-EDTA solution (Cat. #GNM25200, Genom) for further analysis.
HCT-116 cells were subsequently seeded at a density of 2×103 cells per well with the B.a or PBS incubated-CAFs(MOI=1:10) in the 96-well plate for 24, 48,72 hours, then the Cell viability was analyzed using a Cell Counting Kit (CCK-8, Cat. #CK04, Dojindo) according to the manufacturer’s instructions at different time points. Briefly, after removing the medium, cells were incubated with CCK8 for 2 hours and the absorbance was determined at 450 nm, each test was repeated five times.
For LiCl stimulation assay, CAFs were incubated with Wnt signaling agonist lithium chloride (LiCl) (20 mmol/L) for 24 h, then the expression of c-Myc, Cyclin D1 and GAS1 were analyzed.
Data were expressed as mean ± Standard Deviation (SD) and were analyzed by paired or unpaired Student’s t test, one-way ANOVA test, Mann Whitney test or spearman correlation analysis. A P value < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 5.04 software (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 19.0 for Windows (SPSS Inc., Chicago, IL, USA).