DOI: 10.5061/dryad.w0vt4b8tq
title: Response to immune checkpoint blockade improved in pre-clinical model of breast cancer after bariatric surgery

description: Bariatric surgery is becoming more prevalent as a sustainable weight loss
 approach, with vertical sleeve gastrectomy (VSG) being the first line of
 surgical intervention. We and others have shown that obesity exacerbates
 tumor growth while diet-induced weight loss impairs obesity-driven
 progression. It remains unknown how bariatric surgery-induced weight loss
 impacts cancer progression or alters responses to therapy. Using a
 pre-clinical model of diet induced obesity followed by VSG or diet-induced
 weight loss, breast cancer progression and immune checkpoint blockade
 therapy was investigated. Weight loss by bariatric surgery or weight
 matched dietary intervention before tumor engraftment protected against
 obesity-exacerbated tumor progression. However, VSG was not as effective
 as dietary intervention in reducing tumor burden despite achieving similar
 extent of weight and adiposity loss. Circulating leptin did not associate
 with changes in tumor burden. Uniquely, tumors in mice that received VSG
 displayed elevated inflammation and checkpoint ligand PD-L1. Further, mice
 that received VSG had reduced tumor infiltrating T lymphocytes suggesting
 an ineffective anti-tumor microenvironment. VSG-associated elevation of
 PD-L1 prompted us to next investigate the efficacy of immune checkpoint
 inhibitors in lean, obese, and formerly obese mice that lost weight by VSG
 or weight matched controls. While obese mice were resistant to
 immunotherapy, anti-PD-L1 potently impaired tumor progression after VSG
 through improved anti-tumor immunity. Thus, in formerly obese mice,
 surgical weight loss followed by immunotherapy reduced breast cancer
 burden. Further studies are necessary to determine how bariatric surgery
 sensitizes tumors to immune checkpoint inhibition.
description: Mice and diets. Animal studies were performed with approval and
 in accordance with the guidelines of the Institutional Animal Care and Use
 Committee (IACUC) at the University of Tennessee Health Science Center
 (Animal Welfare Assurance Number A3325-01) and in accordance with the
 National Institutes of Health Guide for the Care and Use of Laboratory
 Animals. The protocol was approved under the protocol identifier 21.0224.
 All animals were housed in a temperature-controlled facility with a 12-h
 light/dark cycle and ad libitium access to food and water, except where
 indicated. Three-week-old female C57BL/6J (Jackson stock number: 000664)
 mice were shipped to UTHSC and acclimated 1 week. Four-week-old mice were
 randomized to either obesogenic high fat diet (HFD, D12492i – 60% kcal
 derived from fat) or low fat diet (LFD, D12450Ji- 10% kcal derived from
 fat) from Research Diets Inc. (New Brunswick, NJ) for 16 weeks (age 4
 weeks to 20 weeks old, study design Figure 1A). Mice resistant to diet
 induced obesity (DIO), as defined by less than 28 grams after 16 weeks of
 HFD, were excluded from the study. DIO mice received either a bariatric
 surgery or sham control surgery and dietary intervention as described
 below. Body weight and composition. Body weight was
 measured 2x/week. Body composition including lean mass, fat mass, free
 water content, and total water content of non-anesthetized mice was
 measured weekly using EchoMRI-100 quantitative magnetic resonance whole
 body composition analyzer (Echo Medical Systems, Houston, TX).
 Vertical Sleeve Gastrectomy. To reduce bariatric
 surgery-associated weight loss, peri-operative measures included providing
 liquid diet (Ensure® Original Milk Chocolate Nutrition Shake, Abbott,
 Chicago, IL) and DietGel recovery (Clear H2O, Portland, ME, ID#
 72-06-5022) one day before surgery to all mice. Four hours before surgery,
 solid food was removed to reduce stomach contents. For 4 hours
 pre-surgery, mice were maintained half on half off a heat pad in clean new
 cages. Surgery was performed under isoflurane anesthesia. Vertical sleeve
 gastrectomy (VSG) was performed as previously described [34] with
 additional control dietary intervention for comparison of weight loss
 approaches. The stomach was clamped and the lateral 80% of the stomach was
 removed with scissors. The remaining stomach was sutured with 8-0 to
 create a tubular gastric sleeve. All treatment groups not receiving VSG
 had a sham surgery performed. For sham, an abdominal laparotomy was
 performed with exteriorization of the stomach. Light pressure with forceps
 was applied to the exteriorized stomach. For both VSG and sham surgeries,
 the abdominal wall was closed with 6-0 sutures and skin closed with
 staples. Mice received carprofen (5mg/kg, subcutaneous, once daily) as an
 analgesic immediately prior to and once daily for 3 days following
 surgery. Mice were given 1ml saline at time of surgery. Perioperative
 procedures were performed in accordance with the literature [86, 87]. For
 12 hours post-surgery, mice were maintained half on half off a recovery
 heat pad. Mice were provided Ensure® liquid diet (as above), DietGel
 recovery, and solid food pellets ad libitum for 48 hours post-surgery.
 HFD-fed DIO mice receiving VSG (“HFD-VSG”) were maintained on the same HFD
 for 5 weeks following surgery until euthanasia at study endpoint (Figure
 1A). Control groups that were lean (“LFD-Sham”) or DIO (“HFD-Sham”) were
 maintained on respective LFD or HFD diets following sham surgery. For
 dietary intervention weight loss, DIO mice received sham surgery and were
 subjected to weight loss intervention following sham surgery for 5 weeks
 until endpoint. “Weight Matched” (WM) mice were controls to the HFD-VSG
 mice by weight matching through restricting intake of HFD [88]. On
 average, mice consumed 1.7g (ranging from 1.0-2.5 g or 8.84 kcal (5.2-13.0
 kCal) per day of HFD. Mice were fed at the start of the dark cycle. 78.9%
 of VSG mice survived to endpoint (30/38). Tumor cell
 implantation. E0771 murine adenocarcinoma breast cancer cell line was
 originally isolated from a spontaneous tumor from C57BL/6 mouse. E0771
 cells were purchased from ATCC (CRL-3461) and stable transfected to
 express luciferase (luc) [85] by the Korkaya group at Augusta University
 [52, 85]. Cells were cultured as described previously [52]. Briefly, cells
 were cultured in RPMI containing 10% FBS, 100 UI/mL of penicillin, and 100
 μg/ml streptomycin in a humidified chamber at 37°C under 5% CO2. E0771
 cells were injected in the left fourth mammary fat pad of 22-week-old
 C57BL/6J females at 250,000 cells in 100μl of 75% RPMI / 25% Matrigel.
 When tumors became palpable (typically one week after implantation), tumor
 growth was monitored 2x/week by measuring the length and width of the
 tumor using digital calipers. Tumor volume was calculated using the
 following formula: Volume = (width)2 × (length)/2 [52]. No tumors failed
 to take, and tumor regression was not detected. At the endpoint on day 21
 after tumor cell injection, excised tumor mass was determined.
 Immune checkpoint blockade. In a separate experimental cohort
 limited to HFD-VSG and controls including LFD-Sham, HFD-Sham, and WM-Sham,
 mice were subjected to the same dietary and surgical study design above
 (Figure 1A). After 20 weeks on LFD or HFD, 24-week-old mice received
 either a sham or VSG surgery. Two weeks following surgery, mice were
 injected with E0771-luc cells as above. Immune checkpoint blockade (ICB)
 included anti PD-L1 antibody (Clone 10F.9G2, #BE0101) and IgG2b isotype
 control (Clone LTF-2, #BE0090), purchased from BioXcell (West Lebanon,
 NH). Antibody administration by intraperitoneal (i.p.) injection began
 three days after E0771 cell injection when tumors were palpable (width of
 &gt;2.5mm). Mice were injected every third day for 21 days until
 endpoint (8mg/kg) [89]. Tissue and blood collection.
 Three weeks after tumor implantation (i.e., five weeks after surgery),
 mice were fasted for 4 h and anesthetized. Blood was collected via cardiac
 puncture into EDTA-coated vials. Plasma was separated from other blood
 components by centrifugation at 1200×g for 45 min at 12°C. Mammary tumors,
 tumor adjacent mammary fat pad, unaffected inguinal mammary fat pad, and
 gonadal adipose were weighed and either flash frozen in liquid nitrogen,
 placed into a cassette and formalin-fixed, or digested into a single cell
 suspension for flow cytometry. All frozen samples were stored at −80°C
 until analyzed. Plasma adipokines and cytokines. Plasma
 collected at sacrifice was used for measuring leptin and IL-6 using the
 Milliplex MAP Mouse Metabolic Hormone Magnetic Bead Panel in the Luminex
 MAGPIX system (EMD Millipore, Billerica, MA). Flow
 cytometric analysis of tumors and adjacent mammary adipose tissue. Flow
 cytometry analysis was done as previously described [52]. In brief,
 excised tumors (200 mg) were dissociated in RPMI media containing enzyme
 cocktail mix from the mouse tumor dissociation kit (Miltenyi Biotec,
 Auburn, CA) and placed into gentleMACS dissociators per manufacturer’s
 instructions. Spleen single cell suspensions were obtained by grinding
 spleens against 70μm filter using a syringe plunger. Following red blood
 cell lysis (Millipore Sigma, St. Louis, MO), viability was determined by
 staining with Ghost dye (Tonbo Biosciences Inc.) followed by FcR-blocking
 (Tonbo). Antibodies were titrated, and separation index was calculated
 using FlowJo v. 10 software. Cells were stained with fluorescently labeled
 antibodies and fixed in Perm/fix buffer (Tonbo). Stained cells were
 analyzed using Bio-Rad ZE5 flow cytometer. Fluorescence minus one (FMO)
 stained cells and single color Ultracomp Beads (Invitrogen, Carlsbad CA)
 were used as negative and positive controls, respectively. Data were
 analyzed using FlowJo v 10 software (Treestar, Woodburn, OR). Total immune
 cells from tumor and tumor adjacent mammary fat pad (including tumor
 draining lymph node, TdLN) were gated by plotting forward scatter area
 versus side scatter area, single cells by plotting side scatter height
 versus side scatter area, live cells by plotting side scatter area versus
 Ghost viability dye, and immune cells by plotting CD45 versus Ghost
 viability dye. T cells were gated as follows in tumor CD3+ T cells (CD3+),
 and CD8+ T cells (CD3+, CD8+). Macrophages are gated as CD11b+, F480+.
 Monocytic myeloid derived suppressor cells (M-MDSC) are gated as CD11b+
 Ly6Chigh, Ly6G-. Non-immune cells were gated as CD45- and mean fluorescent
 intensity (MFI) for PD-L1. Gates were defined by FMO stained controls and
 verified by back-gating of cell populations. Gating schema is shown
 supplemental file 2. Flow cytometric analysis of E0771
 breast cancer cells.  E0771-luc cells were treated with recombinant mouse
 IL-6 (200pg/mL) for four hours. Representative biological replicate
 plotted, with N=3 biological replicates with significance. Following
 trypsinization, cells were stained with Ghost dye (Tonbo Biosciences Inc.)
 followed by FcR-blocking (Tonbo) and fluorescent PD-L1 antibody. Flow
 cytometry performed and analyzed as above for PD-L1 MFI.
   RNA sequencing (RNA-seq). mRNA was extracted
 from tumor tissue using RNeasy mini kit (QIAGEN, Germantown, MD) and
 mammary fat pad tissue using a kit specific for lipid rich tissue (Norgen
 Biotek, Ontario, Canada). The integrity of RNA was assessed using Agilent
 Bioanalyzer and samples with RIN &gt;8.0 were used. Libraries were
 constructed using NEBNext® Ultra™ RNA Library Prep Kits (non-directional)
 for Illumina, following manufacturer protocols. mRNA was enriched using
 oligo-dT beads. Libraries were sequenced on NovaSeq 6000 using paired-end
 150 bp reads. There was no PhiX spike-in. Data was analyzed as described
 previously [52, 90]. RNA-seq statistical differences between experimental
 groups were determined as described previously [52]. In brief,
 Benjamini-Hochberg procedure was used to control false discovery rate
 (FDR) for adjusted P value. RNA-seq data has been uploaded as GEO
 GSE174760, GSE174761, and GSE174762. Transcript-level abundance was
 imported into gene-level abundance with the R package tximport. Genes with
 low expression were identified and filtered out from further analysis
 using filterByExpr function of the edgeR package in R software. Voom
 transformation function was applied to normalize log2-cpm values using
 mean-variance trend in the limma software package. ClaNC was used to
 create classifier genes that characterize the groups of interest for
 semi-supervised heatmaps. Database for Annotation, Visualization and
 Integrated Discovery (DAVID) v6.8 was used for pathway analysis [91].
 Immune infiltration estimations based on bulk gene expression data from
 RNA-seq was plotted using TIMER2.0 [92] and cell-type identification
 estimating relative subsets of RNA transcripts (CIBERSORT) [93].
 Bariatric Surgery Patient RNA-seq. Patient gene expression from
 subcutaneous adipose tissue pre- and post- bariatric surgery was
 downloaded from GSE65540 [43] and counts were normalized using counts per
 million (CPM). EdgeR was used for differential expression analysis and
 significance was defined as adjusted p-value of &lt; 0.1.
 Benjamini-Hochberg was used to calculate the FDR. Mouse and human Venn
 diagram was created using the interactive Venn website.
 Gene expression. Total RNA was isolated from tumors and reversed
 transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit
 (Applied Biosystems). qRT-PCR was performed with iTaq Universal SYBR Green
 Supermix (Bio-Rad). Primers span an exon-exon junction and were designed
 with Primer-BLAST (NCBI). Relative gene expression was calculated
 normalized to 18S transcript with 2–∆∆Ct. Primer sequences are:
             Ifng F:GGATGCATTCATGAGTATTGC, Ifng
 R:GTGGACCACTCGGATGAG,             Prf1
 F:GAGAAGACCTATCAGGACCA, Prf1 R:AGCCTGTGGTAAGCATG,
             Gzmb F:CCTCCTGCTACTGCTGAC, Gzmb
 R:GTCAGCACAAAGTCCTCTC,             18S F:
 TTCGGAACTGAGGCCATGATT, 18S R:TTTCGCTCTGGTCCGTCTTG
   Histology and quantification. Tumors and
 normal 4th mammary fat pads, (contralateral to the injected tumor bearing
 mammary fat pad) were isolated at the time of sacrifice and fixed in 10%
 formalin. Formalin fixed paraffin embedded (FFPE) sections from tumors and
 adipose were cut at 5 µm thickness. FFPE sections were stained with
 Hematoxylin and Eosin and scanned by Thermo Fisher (Panoramic 250 Flash
 III, Thermo Fisher, Tewksbury, MA) scanner and adipocyte area of N=50
 adipocytes were quantified using software (Case Viewer) along the longest
 diameter per adipocyte. Statistics. Statistical
 differences between experimental groups were determined using One-way or
 Two-way ANOVA (as noted in figure legends) with Fisher’s LSD test for
 individual comparisons. Outliers were identified and excluded based on the
 ROUT method with Q=1%. For body weight, body composition, and tumor volume
 over time within animals, data was treated as repeated measures. All
 statistics were performed using statistical software within Graphpad Prism
 (Graphpad Software, Inc., La Jolla CA). All data are shown as mean ±
 standard error of the mean (SEM). P values less than 0.05 were considered
 statistically significant. Sample size was determined by power analysis
 calculations and pilot experiments. Group allocation was done to ensure
 equal distribution of starting body weight between groups. Study approval.
 Animal studies were performed with approval and in accordance with the
 guidelines of the Institutional Animal Care and Use Committee (IACUC) at
 the University of Tennessee Health Science Center and in accordance with
 the National Institutes of Health Guide for the Care and Use of Laboratory
 Animals.