Animals
All animal experiments were approved by The Ohio State University Institutional Animal Care and Use Committees and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Anesthesia and euthanasia procedures followed American Veterinary Medical Association guidelines. Two cohorts of nulliparous female 7- to 8-week old Balb/c mice (purchased from Charles River, Wilmington, MA) were housed 5/cage and acclimated to the temperature-controlled (22 ± 1 °C) vivarium with a 14:10 light:dark cycle (lights off at 015:00 h). Rodent chow (Teklad 7912) and water were available ad libitum throughout the study and cotton nestlets and plastic huts were provided for nesting. After 2 weeks of acclimation, mice were pseudorandomly assigned to 3 experimental groups to equalize initial body mass amongst groups: sham surgical control (Control), tumor recipients (Tumor), and tumor recipients that later received tumor resection (Resected) (Fig. 1A). Each animal served as an experimental unit and none of the data collected was excluded. Researchers were not blinded to treatment groups, as monitoring tumor mass was part of humane animal study endpoints. Sample size in each experiment was calculated to ensure 80% power with 0.05 type-I error for the primary analysis derived from our previous studies. Cohort N1 was utilized for food intake and colon barrier function data (Figs. 1A, C and 2, Control n = 19, Tumor n = 11, Resected n = 11), while cohort N2 was utilized for all other data presented (Figs. 3, 4 and 5, Control n = 5, Tumor n = 9, Resected n = 6). 24-h food intake was measured between days 18–19 post tumor induction (Tumor, Control) or tumor resection (Resected). Body mass was taken just prior to animal sacrifice.
Tumor cells
Non-metastatic murine 67NR mammary tumor cell line, which originated from a spontaneous mammary adenocarcinoma in a Balb/c mouse [24] was generously provided by Drs. Fred Miller and Lisa Polin at Karmanos Cancer Institute (Detroit, MI USA). Cells were cultured in Dulbecco’s Modified Eagle Media with 10% FBS, 2 mM L-glutamine, 1 mM nonessential amino acids, and 100U/mL Pennicillin-Streptomycin at 37 °C and 5% CO2.
Tumor induction
To induce mammary tumors, 5 × 106 tumor cells (embedded in Matrigel, phenol red-free, BD Biosciences) were surgically inoculated into the 4th mammary fat pad of mice anesthetized with isoflurane vapors as previously described [25]. Two unique strengths of this mammary tumor model are: 1) the tumors stem from a syngeneic murine cell line, allowing for completely immunocompetent mice, and 2) the surgical intra-mammary fat pad tumor cell inoculation makes the tumor orthotopic. Most mouse models of cancer rely on immunodeficient mice and heterotopic tumors, which greatly limit the translational relevance. Tumors were induced in Resected mice approximately 2 weeks prior to the Tumor mice to allow time for tumor resection and healing prior to tissue collection (see Fig. 1A). Body mass and tumor dimensions (palpable 7–9 d post-induction) were measured 2x/week, and control mice were similarly handled.
Tumor resection
Tumors of the Resected mice were surgically removed using a modified radical mastectomy procedure as previously described [25]. Mice were anesthetized with isoflurane, and tumors were surgically removed along with mammary tissue, fat, and lymph nodes. Buprenorphine (0.05 mg/kg; s.c) was administered immediately after surgery and every 6–12 h post-surgery for 72 h. Sham surgeries were conducted in the control and tumor groups so that each mouse received 2 surgeries to control for effects of anesthesia and wound healing (see Fig. 1A).
Gene expression
Colons were collected and frozen on dry ice for RT-qPCR. Brains were removed, and the frontal cortex -a brain region that regulates mood and cognition- was collected via fresh dissection prior to freezing on dry ice. RNA was isolated from the distal colon and frontal cortex with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA concentration and purity was measured using a NanoDrop™ Spectrophotometer (Thermo Fisher Scientific). Equal amounts of RNA for each sample were reverse transcribed using the qScript® cDNA SuperMix kit (Quantabio) according to manufacturer’s recommendations. Gene expression was measured using TaqMan™ Fast Advanced Master Mix, TaqManTM predesigned gene expression primers/probes (Thermo Fisher Scientific) and amplified using a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific). Expression was quantified using the ΔCt method with Gapdh as the endogenous control.
LBP immunoassay
Mice were euthanized by rapid decapitation and trunk blood was collected using a heparinized Natelson tube. Plasma was separated from whole blood by centrifugation and stored at − 80 °C. Lipopolysaccharide binding protein (LBP) concentration was quantified in plasma using the LBP Mouse ELISA kit (HycultBiotech) according to the manufacturer’s protocol.
Immunohistochemistry
The distal colon was removed, washed with PBS, and fixed in 10% formalin for 24 h. Distal colons were sectioned and stained by the Comparative Pathology and Mouse Phenotyping Shared Resource at The Ohio State University. Heat-induced epitope retrieval was performed in a vegetable steamer for 20 min in pH 6 citrate buffer, and anti-F4/80 antibody (clone MCA497, BioRad Laboratories, Hercules, CA) was utilized as a marker of monocytes and macrophages to assess infiltration into colon tissue.
Fecal Bacteriome sequencing
Two days prior to tissue collection, fecal samples were collected from each mouse and flash frozen on dry ice. Stool samples were sent to The Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory for DNA extraction, library preparation, and high-throughput sequencing. Paired-end (250 nt forward and reverse) sequences of the V4 hypervariable region of the 16S rRNA gene (515F-806R) were generated on the Illumina MiSeq. Quantitative Insights into Microbial Ecology (QIIME) 2.0 [26] was utilized for amplicon processing, quality control with DADA2, and downstream taxonomic assignment using the SILVAv132 database [27]. Sequencing of these samples initially resulted in 1,834,408 paired-end sequences (median = 93,912; maximum = 118,636; minimum = 46,468). After quality control, 1,103,728 high-quality sequences remained (median = 55,832; maximum = 71,371; minimum = 32,520). Samples were rarefied to 32,520 sequences for downstream analyses and no samples were excluded.
Fluorescence in-situ hybridization (FISH)
Spleen and Tumor tissues were fixed in methacarn (60% methanol, 30% chloroform, 10% acetic acid) overnight, embedded in paraffin, and sectioned at 4 μm on positively charged microscope slides. Sections were dewaxed by heating on a heat block at 60 °C for 10 min, two subsequent incubations in xylene substitute at 35 °C and room temperature respectively for 10 min each, and a final incubation in 100% ethanol for 5 min. Then sections were incubated in hybridization buffer (0.9 M NaCl, 0.02 M Tris-HCl, 20% formamide, 10% SDS) for 10 min at 50 °C in a humidified slide chamber in a hybridization chamber, dried and outlined with a PAP pen, then incubated with 0.5 μg of probe EUB (/5Cy3/GCTGCCTCCCGTAGGAGT/3Cy3Sp/) under a cover slip in hybridization buffer in a humidified slide chamber in a hybridization chamber overnight. The next day, sections were washed with washing buffer (0.9 M NaCl, 0.02 M Tris-HCl) twice for 10 min, incubated with DAPI for 15 min, and washed once with PBS for 5 min. Finally, sections were processed for autofluorescence reduction and mounting with the Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Burlingame, CA) according to manufacturer instructions.
Cytokine immunoassay
Spleen and tumor fragments were lysed via sonication (20 s, 25% amplitude) in 150–300 μL of 50 mM Tris-base buffer (0.2 mM PMSF, 100 mM amino-n-caproic acid, 10 mM EDTA, 5 mM benzamidine) plus one cOmplete mini protease inhibitor cocktail tablet per 10 mL of buffer (Millipore Sigma). Samples were centrifuged at 14,000 RPM at 4 °C for 10 min and the supernatant retained. A standard Bradford assay was used to determine the protein concentration of each sample. The concentration of IL-10, IL-1β, IL-6, CXCL1, MCP-1, and TNFα were measured using a multi-plex cytokine array (U-Plex, MesoScale Discovery) according to manufacture protocol. Briefly, 125 μg of tumor protein or 50 μg of spleen protein was incubated overnight at 4 °C while shaking in the antibody-coated, U-PLEX Biomarker Group 1 (ms) Assays, SECTOR plate. The plate was washed, incubated with detection antibody solution, washed, and read immediately following the addition of Read Buffer. The plate was read on a MESO QuickPlex SQ 120 Instrument. The signal CV for each cytokine/chemokine were as follows: IL-1β and TNFα ≤5%, CXCL1 and MCP-1 ≤ 10%, and IL-6 ≤ 15%.
Bacterial culture and identification
Flash-frozen spleen tissue was brought to room temperature in 1 mL PBS, pulverized with autoclaved mortar and pestle, and plated on autoclaved BBL Schaedler agar (BD Biosciences, San Jose, CA). Plates were incubated for 5 days in a humidified incubator at 37 °C with 5% CO2. Individual colonies were then transferred to 5 mL BBL Schaedler broth (BD Biosciences, San Jose, CA) for 5 days under the same incubation conditions. After 5 days, cultures were centrifuged at 5000 g for 10 min, the supernatant was removed, and DNA was isolated with the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to manufacturer instructions.
DNA was quantified using the Qubit high-sensitivity dsDNA quantification assay (ThermoFisher Scientific, Waltham, MA) according to manufacturer instructions, amplified via PCR to produce amplicons of the V3-V5 hypervariable region of the 16S gene (357F-926Rb) [28], cleaned up with DNA Clean and Concentrator-25 (ZYMO Research, Irvine, CA), and sequenced by Eurofins Genomics (Louisville, KY).
Correlation network analysis
All data that were significantly different in either tumor or resected groups relative to control as well as tumor cytokine concentrations were tested for all potential Spearman correlations. Correlations that were significant at p < 0.05 and rho> 0.4 were plotted in a correlation network for analysis via Cytoscape.
Statistical analyses
Results of body and spleen mass, food intake, gene expression, LBP ELISA, F4/80 IHC, MSD, and spleen FISH were tested for normal distribution and equal variances. Assumptions of parametric testing were met, a one-way ANOVA was used, and followed by multiple t-tests to compare between control, tumor, and tumor resected groups. Bacterial 16S rRNA gene amplicon sequencing data was analyzed via PERMANOVA (diversity), and Wilcoxon (genus-level differential abundances) tests. Spleen culture positivity was tested via Chi-square test. Significance was set at p < 0.05.
Data sharing
The data that support the findings of this study are available from the corresponding author upon reasonable request.