Animal experiments
All mouse experiments were performed in accordance with institutional guidelines for animal care and use established in the Principles of Laboratory Animal Care (directive 86/609/EEC). Animal work was only initiated upon approval by the Italian Istituto Superiore di Sanità (ISS) with authorization no. 40/2022, protocol 22418.167 or by the Animal Ethics Committee of the Netherlands Cancer Institute. Six-to-eight-week-old female athymic nude mice (purchased from Envigo) were used in the experiments at the Animal Facility of the IRCCS Ospedale Policlinico San Martino (Genoa). These mice were housed in Sealsafe Plus GM500 individually ventilated cages (IVCs) held on DGM Racks at 22 ± 2 °C and approximately 50–60% relative humidity under a 12 h:12 h light:dark lighting cycle and with food (standard diet, 4RF18, Mucedola) and water ad libitum. Mice were acclimatized for one week before experiments were initiated. To allow MCF7 xenograft growth, a 17β-oestradiol-releasing pellet (Innovative Research of America) was inserted in the intra-scapular subcutaneous region under anaesthesia conditions, the day before cell injection. Xenografts were established by subcutaneous injection of 5 × 106 MCF7 cells to both flanks of the mouse (experiments in Figs. 1a,b, 2 and 4a,b), or orthotopic injection of 3 × 106 MCF7 cells into the fourth abdominal fat pad (experiments in Fig. 4d and Extended Data Fig. 3a,b). Treatment was initiated when the tumours appeared as established palpable masses (~2 weeks after cell injection). In each experiment, mice were randomly assigned to receive one of the following treatments or their combinations, as indicated: control (ad libitum diet); TMX (45 mg kg−1 per day in peanut oil, oral gavage3,39), fasting (water only, for 48 h every week3,40), Dexa (4 mg kg−1 every other day in physiological solution, intraperitoneal41), IGF1 (200 μg kg−1 body weight, intraperitoneal twice a day on the days of fasting); insulin (20 mU kg−1 body weight, intraperitoneal once a day on the days of fasting); leptin (1 mg kg−1 body weight, intraperitoneal once a day on the day of fasting). During the 48 h of fasting, mice were individually housed in a clean, new cage to reduce coprophagy and the intake of the residual chow. Body weight was measured immediately before, during and after fasting. Fasting cycles were repeated every seven days to allow for complete recovery of body weight before a new cycle. The size of the tumours was measured twice a week and tumour volume was calculated using the formula: tumour volume (in mm3) = (w2 × W) × π/6, where w and W are lengths of the minor side and major side (in mm), respectively. The maximum tumour volume that was permitted by our Institutional Animal Care and Use Committee (IACUC) was 1,500 mm3, and in none of the experiments were these limits exceeded. Tumour masses were isolated at the end of the last fasting cycle, weighed, divided into two parts, snap frozen in liquid nitrogen and stored at −80 °C. Ten slices of 50 μm per tumour sample were subsequently utilized for ChIP–seq, proteomics and RNA-seq analyses. Sample size estimation was performed using PS (power and sample size calculation) software (Vanderbilt University). By this approach, we estimated that the number of mice that were assigned to each treatment group would reach a power of 0.85. The type I error probability associated with our tests of the null hypothesis was 0.05. Mice were assigned to the different experimental groups in a random fashion. Operators were unblinded, as blinding during animal experiments was not possible because mice were subject to a specific diet supply and daily treatment.
To establish mammary intraductal cell line-derived xenograft (MIND-CDX) models, T47D cells were intraductally injected as previously described39,42. Specifically, 1 × 106 T47D cells were dissociated to single cells with 0.05% trypsin and injected intraductally into the abdominal/inguinal mammary glands (both sides) of 8-week-old female NSG mice (Jackson Laboratory) with a 34G needle. To ensure stable outgrowth, T47D MIND-CDX mice were supplemented with 17β-oestradiol (Sigma, E2758) via the drinking water at a concentration of 4 µg ml−1 starting 7 days prior to tumour inoculation via intraductal injection. E2 supplementation was maintained throughout the experiment. To establish TSAE1 allograft models, BALB/c mice (Jackson Laboratory) were intraductally injected with of 1 × 104 single cells in PBS as described above (one gland). The IDC186 MIND-PDX model was established from a pre-menopausal Caucasian breast cancer patient, confirmed positive for ERα (95%) and PR (95%) but negative for HER2 (Extended Data Fig. 6h). To generate the PDX model, 5 × 104 single cells in PBS were intraductally injected into one of the abdominal mammary glands of 8-week-old female NSG mice (Jackson Laboratory) and supplemented with 17β-oestradiol (Sigma, E2758) as described above.
The xenograft model cohorts were monitored three times per week and tumours were palpated and measured via calliper in two dimensions. Mice were enrolled into treatment groups when the largest tumour per animal measured 50 mm3 for T47D and IDC186 xenografts and 25 mm3 for TSAE1 allografts, respectively. Mice were randomly allocated into treatment groups and received the following treatments: (1) vehicle treatment (corn oil, daily, oral gavage); (2) TMX (45 mg kg−1 in corn oil, daily, oral gavage); (3) Dexa (4 mg kg−1, 3 times per week, intraperitoneal injection); or (4) TMX plus Dexa. Mice were treated for 28 consecutive days for the TSAE1 and IDC186, 56 days for T47D (with a 1-week treatment break between days 28 and 35), or until the cumulative mammary tumour burden reached a volume of 1,500 mm3 and thus the maximally permitted disease end point. At euthanasia, mammary glands and full female reproductive tracts were collected in formalin, stained against haematoxylin and eosin (H&E) according to routine procedures, and uteri were analysed for histopathological abnormalities. Tumour measurements and post-mortem analysis were performed in blinded fashion. H&E slides were reviewed by a trained pathologist (J.-Y.S.) in a blinded manner. Slides were digitally processed using a PANNORAMIC 1000 whole slide scanner (3DHISTECH) and captured with the Slidescore software (www.slidescore.com).
Clinical studies of FMD in patients undergoing endocrine therapy for HR+ breast cancer
The NCT05748704 trial was conducted at the IRCCS Ospedale Policlinico San Martino (Genoa), between December 2022 and February 2024 and was approved by the Comitato Etico Regione Liguria. This trial consists of a single-arm phase I/II clinical study of a FMD with solid tumours who are candidates to receive active medical or radiotherapy treatment (or with medical treatment or radiotherapy already ongoing). The nutritional intervention consists of a low-calorie diet lasting 5 days and aimed at providing between 800 and 1,000 kcal day−1 (tentatively 10% carbohydrates, 15% proteins and 75% lipids). Throughout the clinical study, patients have received dietary counselling for the intervals between FMD cycles, aiming at providing an appropriate intake of proteins, essential fatty acids, vitamins and minerals43 and have also been invited to perform light/ or moderate daily muscle training to enhance muscle anabolism44. Study primary outcomes were the effects of the FMD regimen on the circulating levels of factors with pro- or anti-oncogenic activity (including insulin, IGF1, IGFBP1, IGFBP3, leptin, adiponectin, IL-6, TNF and IL-1β), as well as the effect of FMD cycles on leukocyte subpopulations with a role in tumour growth control, such as regulatory T cells, myeloid-derived suppressor cells (MDSCs) as well as natural killer (NK) cells, and its stem cell pool (for example, haematopoietic stem cells, endothelial stem cells, mesenchymal stem cells). Additional information on this trial is available at https://clinicaltrials.gov/ct2/show/NCT05748704. Patient serum for subsequent ELISA assays of circulating growth factors and adipokines has been routinely collected before and after the first, sixth and twelfth FMD cycle. Informed consent was obtained from all patients participating in the clinical trial.
The DigesT study (ClinicalTrials.gov ID: NCT03454282) trial was conducted between July 2018 and December 2020 and in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice. The study protocol was approved by the Institutional Review Board (IRB) and the Ethics Committee of Fondazione IRCCS Istituto Nazionale dei Tumori Milan (INT 157/17). All patients provided written informed consent before any study procedures, as well as for the use of clinical and biological data for research purpose. The FMD nutritional intervention consisted in a 5-day, plant-based, calorie-restricted (up to 600 kcal on day 1; up to 300 kcal on days 2, 3, 4 and 5), low-carbohydrate, low-protein, nutritional regimen, as previously published6. Enrolled patients initiated the FMD 12–15 days before surgery, and underwent blood sampling after at least 8 h complete fasting on the morning (08:30 to 10:00) of FMD initiation (pre-FMD), and on the morning of FMD completion (post-FMD). Tumour samples for RNA-seq analyses were obtained diagnostic core biopsies performed at baseline (Pre-) and from matched surgical specimens (Post-). The primary outcomes of the study were to measure the absolute and relative changes in population of peripheral blood mononuclear cells before and after the FMD. Additional information on this trial is available at https://clinicaltrials.gov/ct2/show/NCT03454282.
ChIP–seq
Snap-frozen xenografted tumours were double fixed using 2 mM of disuccinimidyl glutarate diluted in solution A (50 mM Hepes, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 25 min followed by 1% formaldehyde for 20 min, at room temperature. Cells were then lysed and sonicated accordingly to the protocol previously described45, with the difference of have performed 15 cycles of 30 s on, 30 s off in the sonication step (BioRuptor Pico, Diagenode). Obtained nuclear lysates were incubated overnight with 50 μl of protein A coated Dynabeads magnetic beads (10008D, Invitrogen) conjugated with 5 μg of ERα (06-935, Millipore), H3K27ac (39133, Active Motif), GR (12041S, Cell Signaling), PR (8757S, Cell Signaling) or JUN (9165S, Cell Signaling) antibodies. The resulting immunoprecipitated DNA was submitted for library preparation using the KAPA library kit (KK8234, Roche) and subsequently paired-end sequenced on the Illumina NovaSeq 6000 system with read length of 51 bp. ChIP–seq analyses were performed using an in house pipeline publicly available at https://github.com/sebastian-gregoricchio/ChIP_Zwart (v.0.1.2) with default parameters. In brief, all samples were aligned to reference genome Hg38/GRCh38 using Burrows-Wheeler Aligner46 (BWA v.0.5.10). Reads were filtered based on mapping quality (MAPQ ≥ 20), and duplicate reads were marked with Picard MarkDuplicates (v.2.19.0). MACS2 (v.2.1.2) was used to perform peak calling over input ChIP–seq samples; only peaks with a q-value < 0.01 were retained. DeepTools47 (v.2.5.3) was used to calculate the fraction of reads in peaks (FRiP) and normalized ChIP–seq signal. For visualization purposes, Reads Per Genomic Content (RPGC) normalization (1× coverage) signal was averaged among the replicates per each condition using deeptools bigwigCompare. Genome browser snapshots were generated using the R v.4.0.3 environment and Rseb48 (v.0.3.1) (https://github.com/sebastian-gregoricchio/Rseb). Tornado plots were generated using deepTools (v.2.5.3). Differential peak analyses were performed using diffBind49 (v.3.0.15). Peaks were defined as differential when the |log 2 (fold change)| >1.5 and adjusted P value <0.05. Genomic location annotation of the peaks was performed using ChIPSeeker50 (v.1.26.2) defining the promoter region as −2 kb:transcription start site:+1 kb. Transcription factor binding enrichment from public available datasets—GIGGLE analyses51—were performed using the tool available at the of CistromeDB website (http://cistrome.org/db/).
Cell lines
... continue reading