The gut bacterium, B. pullicaecorum (BCRC-81109), was purchased from the Bioresource Collection and Research Center and was grown in a modified peptone yeast extract broth (PY-X broth: 0.5 g peptone from meat; 0.5 g trypticase peptone; 1.0 g yeast extract; 0.1 mg resazurin; 50 mg L-cysteine-HCI.H₂O; 0.5 g D-glucose; 10 mg CaCl₂.2H₂O; 20 mg MgS0₄.7H₂O; 40 mg K₂HP0₄; 40 mg KH₂P0₄; 0.4 g NaHC0₃; 0.8 mg NaCl; 0.5% xylan in 100 ml distilled water; pH 7.0; DSMZ GmbH) for 3 days under anaerobic conditions at 37°C. The conditioned medium from B. pullicaecorum cultures was harvested by centrifugation (1,000 × g, 15 min) at room temperature to remove the bacteria and sterilized by filtration with a 0.22 µm syringe filter (Thermo Fisher Scientific, Inc.).

BALB/cByJNarl male mice (age, 4–6 weeks; weight range, 20–25 g) were provided by the National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan. All animal experiments complied with the Animal Research: Reporting of In Vivo Experiments guidelines (30). All protocols were approved by the Institutional Animal Care and Use Committees at Cathay General Hospital, Taipei, Taiwan and followed the ‘3Rs’ (Reduction, Refinement and Replacement) (31). All efforts were made to minimize the number of animals and their suffering. The body weight of mice was monitored every week and mice were euthanized when they exhibited weakness and rapid weight loss (15–20% of their body weight) within a few days (32). A total of 3–5 were housed per plastic cage under pathogen-free conditions (humidity, 50±10%; light/dark cycle, 12 h; temperature, 23±2°C) in an individually ventilated cage rack system (Tecniplast S.p.A).

To evaluate the effect of B. pullicaecorum (3.125×10⁷ colony-forming units in 100 µl) on CRC formation, CRC was induced with DMH (40 mg/kg; cat. no. D0741; TCI America; Tokyo Chemical Industry)/DSS (2%; cat. no. D5144; TCI America; Tokyo Chemical Industry), as reported by De Robertis et al (33) and as described in the legend of Fig. 1A. A total of 17 mice were used and randomly divided into the following groups to study the induction of CRC by DMH and the in vivo effect of B. pullicaecorum: i) Control group (CG; n=5), no DMH treatment or B. pullicaecorum administration; ii) DMH mice (n=6), administered DMH by intraperitoneal injection and DSS in drinking water and no B. pullicaecorum administration; and iii) DMH/B. pullicaecorum (BP) mice (n=6), administered DMH, DSS, and B. pullicaecorum by oral gavage. Anuses of mice were imaged on days 29, 32 and 36 following initial B. pullicaecorum administration. Mice were sacrificed 8–9 weeks later with CO₂ euthanasia in a cage. The CO₂ flow rate was set to displace 30% of the cage volume/minute. The following parameters were used to confirm death: i) immobility; and ii) lack of spontaneous breathing for >2 min. During the experiments, the clinical characteristics of mice, including anal bleeding, body weight and serum carcinoembryonic antigen (CEA) levels were recorded.

SW480 colon cancer cell line (ATCC CRL-1459; AJCC stage II) and SW620 lymph node metastatic derivative cell line (ATCC CRL-1831; AJCC stage III) from the same patient were purchased from American Type Culture Collection. The two cell lines were expanded in complete medium [Leibovitz's L-15 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (NQBB International Biological Co., Ltd.)] under 100% atmospheric air (without CO₂) in a 37°C humidified incubator. To evaluate the effects of B. pullicaecorum on CRC cell proliferation, the numbers of SW480 and SW620 cells were respectively counted at days 1 and 5. Briefly, cells incubated with or without 10% conditioned medium from B. pullicaecorum cultures in complete medium were imaged under an Olympus BX41 light microscope (Olympus Corporation) and analyzed using ImageJ software (version 1.52; National Institutes of Health) (34).

Total RNA from SW480 and SW620 cells, which were untreated or treated with 5 mM sodium butyrate (NaB) for 48 h as reported in our previous study (21) was independently extracted using a RNeasy Mini kit (Qiagen, Inc.), according to the manufacturer's protocol. Total RNA (1 µM) was reverse-transcribed to single-stranded cDNA using a high-capacity cDNA Reverse Transcription kit (cat. no. 4368813; Thermo Fisher Scientific, Inc.), as previously described (35). To quantify the expression of GPR43, the reaction mixture containing the cDNA sample, QuantiTect SYBR-Green PCR Master mix (Qiagen GmbH), QuantiTect Primer assay (cat. no. Hs_FFAR2_1_SG; Qiagen GmbH) and RNase-free water was amplified using the following cycling program: 10 min at 95°C, followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. To quantify the expression of SLC5A8, the reaction mixture containing forward (5′-TCTTCCTCCCGGTGTTCTAC-3′) and reverse primers (5′-GAACACATTTGTTAAATCGAAGTTCT-3′), no. 48 universal probe (Roche Diagnostics GmbH), LightCycler TaqMan Master (Roche Diagnostics GmbH) and RNase-free water was amplified by a program (10 min at 95°C, proceeding with 60 cycles at 95°C for 10 sec and at 60°C for 20 sec). The expression of GAPDH (no. 60 universal probe (Roche Diagnostics GmbH); forward primer, 5′-CTCTGCTCCTCCTGTTCGAC-3′; reverse primer, 5′-ACGACCAAATCCGTTGACTC-3′) was used as an internal control for calibration. For control purposes, the human reference cDNA (Clontech Laboratories) was used as a positive control to estimate the relative expression levels in the cells and the non-template reaction was a negative control. All RT-PCR reactions were run in a LightCycler 96 (Roche Diagnostics GmbH) and the data were analyzed using the 2^−ΔΔCq method (36).

Colon tissues were obtained immediately following sacrifice and death and embedded in paraffin. Hematoxylin and eosin (H&E) were added to the paraffin section (thickness, 3–5 µm) to identify non-CRC and CRC areas using the method reported by Fischer et al (37). Immunohistochemistry (IHC) was performed as described by Huang et al (9). Briefly, colon tissue sections were hybridized with anti-GPR43 (1:100 in blocking solution; cat. no. BS-13536R) and anti-SLC5A8 (1:100 in blocking solution; cat. no. BS-6106R) polyclonal antibodies obtained from Bioss Antibodies. GPR43 and SLC5A8 were visualized using 3,3¢-diaminobenzidine (Vector Laboratories, Inc.) as the substrate. A pathologist viewed and categorized the H&E and IHC-stained sections.

Blood samples (100 µl) were collected from a tail incision in each mouse. Sample was centrifuged at 1,000 × g for 15 min at room temperature to isolate the serum. Serum CEA levels were measured using a commercial ELISA kit (cat. no. E-EL-M0232; Elabscience), according to the manufacturer's protocol.

One-way ANOVA with Fisher's least significant difference (LSD) post-hoc test was performed to identify significant differences between groups. Differences in gene expression were compared using Student's t-test. Statistical analyses were performed using the SPSS statistics software (version 22.0; IBM Corp.). Data for cell numbers, body weight and serum CEA levels are presented for a >3 mice or ≥3 experiments with similar results. Data are presented as mean ± SEM. P<0.05 was considered to indicate a statistically significant difference.

Fig. 1A presented the timing of DMH and DSS induction of CRC and B. pullicaecorum administration. Numerous irregular and different sized crypt foci were observed in the inner layer of the colon of the DMH group, particularly in the distal colon and near the rectum (Fig. 1B). The histopathological examination demonstrated no foci in the colons of CG mice and the intestinal tract had an intact mucosal epithelium without any significant dysplastic changes (Fig. 1C, top panel). Conversely, aberrant crypt foci and multiple exophytic tumors in the mucosal layer were observed in the intestinal tract of DMH mice (Fig. 1C, bottom panel).

Visualization at higher magnification detected no specific histological changes in the intestinal mucosa, submucosa or muscular layers of CG mice (Fig. 1C, left middle panel). Furthermore, the neoplastic glands of DMH mice exhibited a complex or fused glandular pattern and notable nuclear atypia (Fig. 1C, right middle panel).

Fig. 1D demonstrated the regressive effect of B. pullicaecorum administration on DMH-induced CRC. In DMH/BP mice, tumors appeared to be downstaged histopathologically. Invasive carcinoma was limited to the submucosa with narrow maximal tumor invasion depth and fewer inflammatory cells infiltrates were observed in the periglandular area. The colonic tumors of DMH mice exhibited an early stage of the carcinogenesis process, which ranged from in situ to minimally invasive adenocarcinoma.

Levels of SCFA transporter (SLC5A8) and receptor (GPR43) were examined by IHC staining of the colon of mice. As shown in Fig. 2, SLC5A8 and GPR43 were detected mainly in the superficial portion of the crypt glands in CG mice, while faint and randomly distributed reactivity was observed in DMH mice. Notably, transporters and receptors were diffusely and moderately to strongly expressed in neoplastic epithelial cells in DMH/BP mice. In addition to the epithelial cell populations, patchy areas with variable cytoplasmic staining for SLC5A8 and GPR43 were observed in the underlying muscular layer.

Phenotypic abnormalities and clinical characteristics of DMH and DMH/BP mice were analyzed. Compared with CG mice, anal bleeding was worse in the DMH mice; however, this was improved in DMH/BP mice (Fig. 3). Body weights and serum CEA levels of mice were measured. As shown in Fig. 4A, the mean body weight increased significantly from 19.2 to 30.0 g in 8 weeks in CG mice and no mice died during this time. Mice in the DMH/BP group exhibited significant weight loss, which was previously reported by Kim et al (38). The rate of weight gain was slower in mice in the groups administered DMH (DMH and DMH/BP mice) compared with CG mice. DMH mice (initial n=6, two died during the experiment) exhibited the lowest weight. DMH/BP mice (initial n=6, one died during the experiment) lost less weight than DMH mice. Briefly, statistical significance was observed at week 7 (P<0.01) and near significance was observed at week 8 (P=0.054) when comparing the body weights of DMH mice and DMH/BP mice. Overall, three mice (two DMH mice and one DMH/BP mouse) died during the experiments due to colon tumors. The rest of the mice were euthanized at the humane endpoint. DMH/BP mice exhibited longer survival rates (83.3±5.2%; Fig. S1), reduced body weight loss and lower serum CEA levels compared with DMH mice (Fig. 4B).

Gas chromatography-mass spectrometry analysis demonstrated that 3.05 mM butyrate was the predominant metabolite in the medium following cultivation of B. pullicaecorum (Table SI). The changes in SW480 and SW620 cells following treatment with the butyrate-containing supernatant from B. pullicaecorum cultures were evaluated using ImageJ software (Fig. 5A). On day 5, SW480 and SW620 cell numbers were lower in cells cultured in 90% cell growth medium + 10% filter-sterilized conditioned medium compared with cells cultured with the control medium (90% cell growth medium + 10% PY-X broth). However, the growth of SW620 cells was more sensitive than SW480 cells to cultivation with 10% filter-sterilized conditioned medium. In the cultures of SW480 and SW620 cells with 10% supernatant from B. pullicaecorum, SW620 cell growth was reduced to 50.7% (from 359,000 cells to 182,000 cells) and SW480 cells to 77.4% (from 159,000 cells to 123,000 cells). This inhibitory effect on the CRC cell growth following butyrate treatment was also reported in our previous study (21). SLC5A8 and GPR43 expressions in butyrate-treated CRC cells were quantified (Fig. 5B and C for SLC5A8 and GPR43, respectively). In comparison with untreated cells, the NaB-treated SW480 cells only presented higher GPR43 (P<0.05), but the NaB-treated SW620 cells expressed the most SLC5A8 (P<0.01) and GPR43 (P<0.05).