GoKawiilCell lines and culture conditions
HKP1 cells were provided by V. Mittal. YUMM1.7 and YUMMER1.7 were provided by I. Watson. HKP1, YUMM1.7 and YUMMER1.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin and streptomycin. MCA205 cells were provided by J. Stagg and were cultured in RPMI-1640 medium containing 10% FBS, 2 mM l-glutamine and 100 UI ml−1 penicillin and streptomycin. Cells were cultured at 37 °C in the presence of 5% CO 2 . Cell lines were not authenticated and were routinely tested for mycoplasma contamination.
Animal ethics statement
All animal experiments and data collection were conducted either at the Goodman Cancer Institute (GCI) at McGill University, or the Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM). Unless indicated otherwise, tumour assays were monitored for 15–24 days, with early humane end-points, including development of tumour ulceration, total volume exceeding 2.5 cm3, or onset of adverse clinical signs. All procedures were approved by respective institutional animal care committees, including the Comparative Medicine and Animal Resources Centre (CMARC, McGill) or the Institutional Animal Care Committee (IACC, CRCHUM), and experiments adhered to approved humane end-points. All experiments complied with the Canadian Council on Animal Care guidelines.
Standard animal housing
Mice were housed in cages with micro-barrier tops (Allentown) on a standard rack system under specific pathogen-free conditions. Each cage contained up to five mice with corn bedding (Envigo), enrichment (Shepherd), irradiated standard chow in the cage-top (Envigo), and autoclaved water. The animal facility maintained a 12 h:12 h light:dark cycle (lights on at 07:00). The following procedures took place in the morning, within a 2 h window across cohorts: (1) non-terminal blood collection for serum and flow cytometry; (2) all dissections; and (3) fasting for GTT. The following procedures took place in the afternoon, within a 3 h window: (1) body weight; (2) stool collections; (3) tumour injections; (4) anti-PD-1 and IgG treatments and tumour measurements; (5) GTT; (6) EchoMRI; and (7) FMT. Note that germ-free experiments were housed under non-standard conditions (see ‘In vivo experimental models’ for additional detail).
Diets
All diets were designed and produced in collaboration with Research Diets. All diets were irradiated by Research Diets and administered ad libitum. The Mediterranean (powdered), Japanese (powdered) and Ketogenic (paste) diets were provided in special feeders. All other diets were in the form of standard pellets delivered in the cage-top and replenished as needed. The Japanese, Mediterranean, Vegan, American, Aspartame and Ketogenic diets were designed to mimic human dietary patterns in terms of ingredient profiles, macronutrient content, fibre sources and fatty acid ratios (Supplementary Tables 1 and 2). Carbohydrate sources include corn starch, maltodextrin, wheat starch, rice starch, potato starch, sucrose and fructose. The Vegan diet contains spinach, broccoli, apple, banana, blueberry, carrot, raspberry and tomato powder (Nubeleaf). Protein sources include beef protein, casein, soy protein, egg white protein and fish protein. Fat sources include soybean oil, corn oil, menhaden oil, palm kernel oil, butter, lard and flaxseed oil. The modified fibre diets were based on the Low Fat diet, with cellulose exchanged for the different fibre source (inulin, pectin and psyllium). The germ-free mice were fed double-irradiated High Fat or Psyllium diets with 1.5× vitamin supplementation. Proportions of ingredients and macronutrient breakdown can be found in Supplementary Tables 1 and 2.
In vivo experimental models
1. 15-week diet model. 15-week diet experiments were performed at the GCI with 4-week-old male C57BL/6J mice purchased from Jackson Laboratories. Upon arrival, mice were fed the standard chow for 1 week during the acclimatization period and were then switched to a special diet for 15 weeks (ad libitum). At 20 weeks of age, mice were injected subcutaneously with 500,000 HKP1 tumour cells in a suspension of 1:1 PBS:Matrigel (Corning). For ICI trials, when tumours were palpable, mice were randomized on a per-cage basis to anti-PD-1 (clone J43, Bio X Cell) or IgG (non-reactive polyclonal Armenian hamster, Bio X Cell), administered intraperitoneally every 3 days until the end-point (16–24 days). Weight-adjusted dosing was used to account for differences in body weight and blood volume (10 mg kg−1 per mouse; Supplementary Table 10). Diet was continued for the duration of the experiment and tumours were monitored by manual calliper. ‘Response’ was defined as a statistically significant reduction in tumour volume in anti-PD-1-treated mice compared with IgG controls, whereas ‘resistance’ was defined as a lack of therapeutic benefit (that is, distinct from hyperprogression, which was not observed in our models). 2. 3-week diet model: 3-week diet experiments were performed at the GCI with 12- to 15-week-old male C57BL/6J mice purchased from Jackson Laboratories (age-matched to the 15-week diet model). Upon arrival, mice were fed the standard chow for 1 week during the acclimatization period and were then switched to a special diet for 3 weeks (ad libitum). Mice were then injected subcutaneously with 500,000 HKP1, YUMM1.7 or YUMMER1.7 tumour cells in a suspension of 1:1 PBS:Matrigel (Corning). When tumours were palpable, mice were randomized on a per-cage basis to anti-PD-1 (clone J43, Bio X Cell) or IgG (non-reactive polyclonal Armenian hamster, Bio X Cell), at a weight-adjusted dose (10 mg kg−1) administered intraperitoneally every 3 days until the end-point (16–24 days). Diet was continued for the duration of the experiment and tumours were monitored by manual calliper. 3. Diet switch model: Diet switch experiments were performed at the GCI with 15-week-old male C57BL/6J mice purchased from Jackson Laboratories. Upon arrival, mice were fed the standard chow for 1 week during the acclimatization period and were then switched to either the High Fat diet or Psyllium diet for 3 weeks (ad libitum). After three weeks, mice were injected subcutaneously with 500,000 HKP1 tumour cells in a suspension of 1:1 PBS:Matrigel (Corning). When tumours were palpable, mice were randomized on a per-cage basis to anti-PD-1 (clone J43, Bio X Cell) or IgG (non-reactive polyclonal Armenian hamster, Bio X Cell), at a weight-adjusted dose (10 mg kg−1) administered intraperitoneally every 3 days until the end-point (21 days). Two days before the first treatment, the High Fat diet was switched to the Psyllium diet, and vice versa. The new diet was continued for the duration of the experiment and tumours were monitored by manual calliper. 4. Antibiotic treatment (sustained during ICI treatment): Long-term antibiotic treatment experiments were performed at the GCI with 4-week-old male C57BL/6J mice purchased from Jackson Laboratories. Upon arrival, mice were fed the standard chow for 1 week during the acclimatization period and were then switched to a special diet for 15 weeks (ad libitum). After 12 weeks of being fed the Low Fat or High Fat diet, mice were administered antibiotics (1 mg ml−1 ampicillin (Sigma-Aldrich), 1 mg ml−1 neomycin (Fisher Scientific), 1 mg ml−1 gentamicin (Sigma-Aldrich), 1 mg ml−1 metronidazole (Sigma-Aldrich), 0.5 mg ml−1 vancomycin (Fisher Scientific)) in sterile drinking water ad libitum for the remainder of the experiment. The solution was replenished every 3–4 days, and bottles were changed weekly. After 15 weeks of special diet and 3 weeks of antibiotics, mice were injected subcutaneously with 500,000 HKP1 tumour cells in a suspension of 1:1 PBS:Matrigel (Corning). When tumours were palpable, mice were randomized on a per-cage basis to anti-PD-1 (clone J43, Bio X Cell) or IgG (non-reactive polyclonal Armenian hamster, Bio X Cell), at a weight-adjusted dose (10 mg kg−1) administered intraperitoneally every 3 days until the end-point (18 days). Tumours were monitored by manual calliper. 5. Mouse-to-mouse FMT: Mouse-to-mouse FMT experiments were performed at the GCI with 15-week-old male C57BL/6J mice purchased from Jackson Laboratories. Mice were fed either a High Fat diet or Psyllium diet for the entire trial, beginning one week before antibiotics. Mice were administered antibiotics (1 mg ml−1 ampicillin (Sigma-Aldrich), 1 mg ml−1 neomycin (Fisher Scientific), 1 mg ml−1 gentamicin (Sigma-Aldrich), 1 mg ml−1 metronidazole (Sigma-Aldrich), 0.5 mg ml−1 vancomycin (Fisher Scientific)) in sterile drinking water ad libitum for 14 days. The solution was replenished every 3–4 days, and bottles were changed weekly. Two days after the mice were switched to regular autoclaved drinking water, fecal samples were collected from donor mice consuming the Psyllium diet and homogenized at a concentration of 1 fecal sample per ml of reduced PBS. One-hundred microlitres of suspension was used for oral gavage of each recipient mouse. Approximately 100 µl of solution was applied to the fur of each mouse. No-FMT control mice received 100 µl of PBS via oral gavage. This process was repeated weekly for the duration of each trial, for a total of five FMTs. 24 h after the second FMT, mice were injected subcutaneously with 500,000 HKP1 tumour cells in a suspension of 1:1 PBS:Matrigel (Corning). When tumours were palpable, mice were randomized on a per-cage basis to anti-PD-1 (clone J43, Bio X Cell) or IgG (non-reactive polyclonal Armenian hamster, Bio X Cell) at a weight-adjusted dose (10 mg kg−1) administered intraperitoneally every 3 days until the end-point (21 days). Tumours were monitored by manual calliper. 6. Human-to-mouse FMT from high-BMI and low-BMI donors: Human-to-mouse FMTs were performed at the CRCHUM, with ethics approval for human sample collection (ethics no. C23046BR) and informed consent from patients. After 48 h of acclimatization, 6-week-old female wildtype C57BL/6J specific pathogen-free mice from Charles River received 3 days of antibiotics solution containing ampicillin (1 mg ml−1), streptomycin (5 mg ml−1) and colistin (1 mg ml−1; Sigma-Aldrich) in sterile drinking water and were fed standard chow. FMT, using faeces from patients with NSCLC (Supplementary Table 9), was performed by thawing fecal material, and 200 µl of the suspension (100 mg ml−1) was then transferred by oral gavage. An additional 100 µl was applied on the fur of each animal. Two weeks after FMT, mice were implanted subcutaneously with 800,000 MCA205 cells. When tumours were palpable, mice were randomized to anti-PD-1 (clone RMPI-14, Bio X Cell) or IgG (clone 2A3, Bio X Cell) at a fixed dose of 250 µg per mouse intraperitoneally every 3 days. Tumour growth was monitored by manual calliper. 7. Human-to-mouse FMT from ICI-refractory patient: Human-to-mouse FMT was performed at the CRCHUM, using a human fecal sample (Supplementary Table 3) from the CHUM lung cancer biobank. Appropriate ethics approval was obtained at the CRCHUM in Montreal (ethics 17.035), with informed consent from the patient. After 48 h of acclimatization, 6-week-old female wildtype C57BL/6J specific pathogen-free mice from Charles River were fed the High Fat diet or the Psyllium diet with ad libitum access for the duration of the trial. 10 days after the start of the trial, an FMT using the patient’s faeces was performed by thawing fecal material, and 200 µl of the suspension (100 mg ml−1) was then transferred by oral gavage. An additional 100 µl was applied on the fur of each animal. Two weeks after FMT, mice were injected subcutaneously with 500,000 HKP1 cells. When tumours were palpable, mice were randomized to anti-PD-1 (clone RMPI-14, Bio X Cell) or IgG (clone 2A3, Bio X Cell) at a fixed dose of 250 µg per mouse intraperitoneally every 3 days. Tumour growth was monitored by manual calliper. 8. Single-strain supplementation in specific pathogen-free mice: Single-strain supplementation experiments were performed at the CRCHUM. After 48 h of acclimatization, 6-week-old female wildtype C57BL/6J mice from Charles River were fed the High Fat diet ad libitum. After 11 days, mice received 3 days of antibiotics solution in sterile drinking water, containing ampicillin (1 mg ml−1), streptomycin (5 mg ml−1) and colistin (1 mg ml−1; Sigma-Aldrich). Mice were then injected subcutaneously with 500,000 HKP1 cells (resuspended in 1:1 Matrigel:PBS). Beginning on the day of tumour cell injection, oral gavage of 100 µl of bacterial suspension (L. johnsonii or M. gordoncarteri) or PBS were performed every 3 days until the end-point. When tumours were palpable, mice were randomized to anti-PD-1 (clone RMPI-14, Bio X Cell) or IgG (clone 2A3, Bio X Cell) at a fixed dose of 250 µg per mouse intraperitoneally every 3 days. Tumour growth was monitored by manual calliper. 9. Monocolonization in germ-free mice: Germ-free 7- to 10-week-old female C57BL/6J mice were purchased from the International Microbiome Centre Germ-Free Facility (University of Calgary) and maintained at the CRCHUM germ-free animal facility. Mice were housed in sterile individual ventilated cages at 21–23 °C, 40–60% humidity on a 12 h:12 h light:dark cycle. Mice had free access to water and food (Teklad Global 2918). Mice were housed in sealed positive pressure IVC (Sentry SPP) in an exclusive holding room with the same environmental parameters described above. After 48 h of acclimatization, mice were fed double-irradiated High Fat diet (D12492-1.5V, Research Diets) or Psyllium diet (D21021104B-1.5V, Research Diets) with ad libitum access to food. After 2 weeks, mice were implanted with 500,000 HKP1 cells or 800,000 MCA205 cells (resuspended in 1:1 Matrigel:PBS). Beginning on the day of tumour cell injection, an oral gavage of 200 µl of bacterial suspension (L. johnsonii or M. gordoncarteri at OD 1) or PBS were performed every 3 days until the end-point. When tumours were palpable, mice were treated with anti-PD-1 (clone RMPI-14, Bio X Cell) or IgG (clone 2A3, Bio X Cell) at a fixed dose of 250 µg per mouse intraperitoneally every 3 days. Tumour sizes were measured with a manual calliper. The tumour cells were manipulated in a clean environment under a biological hood and tested negative for mycoplasma. The tubes containing the tumour cells, the bacteria suspension, and the anti-PD-1 or isotype control were sterilized with a cold sterilant and directly transferred under the hood where the mice were manipulated. The callipers were first sterilized in an autoclave at 132 °C for 20 min. At the time of the experiment, a second sterilization was performed, during which the sterilization pouches containing the callipers were sterilized with a cold sterilant and directly transferred under the hood where the mice were manipulated. After the measurement was performed, the calliper was placed on the steel grid inside the cage to remain sterile for subsequent measurements. 10. DAT supplementation in vivo: DAT supplementation experiments were performed at the GCI with 11-week-old male C57BL/6J mice from the in-house colony. Sterilized DAT drinking water solution was prepared using 100 mM 3-(4-Hydroxyphenyl)-propionic acid (DAT, Sigma-Aldrich) in autoclaved water neutralized with NaOH. Following the 3-week tumour model with the Psyllium diet, mice were administered DAT-supplemented drinking water beginning on the day of tumour injection. Mice were injected subcutaneously with 500,000 HKP1 tumour cells in a suspension of 1:1 PBS:Matrigel (Corning). When tumours were palpable, mice were randomized on a per-cage basis to anti-PD-1 (clone J43, Bio X Cell) or IgG (non-reactive polyclonal Armenian hamster, Bio X Cell), at a weight-adjusted dose (10 mg kg−1) administered intraperitoneally every 3 days until the end-point (day 20). Diet and DAT-water were continued for the duration of the experiment and tumours were monitored by manual calliper.
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