DNA
Sequences encoding 4ab, P28ζ, PTr or GLUT5 were cloned downstream of the human EF1a into the lentiviral transfer vector pUltra (Addgene plasmid # 24129). Construction of DNA vectors was conducted using NEBuilder HiFi DNA Assembly Kit as per manufacturer instructions (NEB). Assembly reactions were transformed by mixing the entire reaction with 25 mL of NEB 5-alpha competent E. coli (NEB) as per manufacturer instructions. Ampicillin resistant bacterial colonies were transferred to 15 mL of LB broth containing ampicillin (Sigma) (100 mg/mL) and grown at 37°C for 18 hr at 220 RPM. Plasmid DNA was extracted from bacterial cultures with E.Z.N.A Plasmid DNA Mini Kit II (Omega Bio-Tek) as per manufacturer instructions.
Cell Lines
HEK-293T, PC3-LN3, PC3-LN3-PSMA and MCF7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% Foetal Bovine Serum (FBS) (Sigma). In experiments where media containing different concentrations of glucose or alterative to glucose are used, PC3-LN3-PSMA or MCF7 cells were cultured in glucose-free DMEM supplemented with 10% FBS. All cell lines were cultured at 37°C in 5% CO2.
Lentiviral Production in HEK-293T
1x107 HEK-293T cells were plated in 150 mm dishes and allowed to adhere for 24 hrs. HEK-293T cells were then transfected with 25 mg of lentiviral transfer plasmid, 12.5 mg pVSV-G and 12.5 mg of pCRV-1 (kind gifts from Dr Caroline Hull, KCL). Transfection reactions were prepared by mixing plasmids with 150 mg of polyethylenimine (Sigma) in 1 mL of serum-free DMEM and incubating at room temperature for 25 mins before reactions were added dropwise to cells. 24 hrs after transfection, media was aspirated and replaced with 17.5 mL of fresh media. 48 hrs after transfection, virus-containing media was removed and transferred to a 50 mL conical tube which was stored at 4°C. 17.5 mL of fresh media was added to cells. 72 hrs after transfection, virus-containing media was again removed from cells and pooled with the virus-containing media removed from cells the previous day.
Lentivirus was then concentrated from virus-containing media by first centrifuging at 1500 g for 5 min to remove detached cells and debris. The supernatant containing lentivirus was transferred to a new 50 mL conical tube and centrifuged at 20000 g for 90 mins. The supernatant was aspirated, and the virus-containing pellet was resuspended in 200 mL of phosphate-buffered saline (PBS). Concentrated lentivirus was stored at -80°C until used.
Isolation, culture, transduction and expansion of human PBMCs
Whole blood was obtained from healthy human donors, after written informed consent, under the approval of the Guy’s and St Thomas’ Research Ethics Committee (reference 09/H0804/92). PBMCs were purified by carefully layering whole blood over 15 mL of Ficoll- Paque (GR Healthcare), before centrifugation at 800 g for 35 min with brake and accelerator set to minimum. PBMCs were then transferred to a clean 50 mL tube and washed twice in PBS, before being resuspended in an appropriate volume of media.
PBMCs were cultured in RPMI 1640 (Gibco) supplemented with 5% human AB serum (Sigma). PBMCs were counted and diluted to 1x106 cells/mL and activated with 5 µg/mL Phytohemagglutinin-L (Sigma)in a 96-well plate. 24 hrs after activation, cultures were supplemented with 100 U/mL recombinant human IL-2 (rhIL-2) (R&D Systems) and 50 µL of concentrated lentivirus was then added to cells and mixed by gently pipetting. For untransduced cells, 50 mL of PBS was added in place of lentivirus.
48 hrs after transduction, PBMCs were washed by adding 1 mL of PBS and centrifuged at 800 g for 5 mins. PBMCs were resuspended in 500 mL of fresh media supplemented with 100 U/mL rhIL-2. For PBMCs transduced with constructs containing the chimeric cytokine receptor 4ab, cultures were supplemented with 3 ng/mL recombinant human IL-4 (rhIL-4) instead of rhIL-2. Cells were then expanded to sufficient numbers by diluting with fresh media and supplementation of cultures with 100 U/mL rIL-2 or 3 ng/mL rIL-4 every 2–3 days.
For metabolite determination experiments, 0.5ml of PBMCs at 1x106 cells/mL were activated with PMA (50 ng/mL) and ionomycin (0.5 mg/mL) (both from Sigma) in 48-well tissue culture plates and incubated for the indicated period of time.
Co-culture of tumour cells lines with PBMCs
PC3-LN3-PSMA or MCF7 cells were plated in either a 96-well, 24-well or 6-well tissue culture plate at a density of 1x105 cells/cm2. Cells were allowed to adhere for 24 hrs. Expanded PBMC were then counted, washed in excess PBS to remove all cytokine and resuspended in fresh media. PBMC were then added to wells containing PC3-LN3-PSMA or MCF7 cells at the indicated E:T ratio and incubated for the indicated time period.
In experiments where defined concentrations of glucose or fructose were used, PC3-LN3-PSMA or MCF7 cells were first plated in standard media and allowed to adhere overnight. Before the addition of PBMC, media was aspirated from PC3-LN3-PSMA or MCF7 cells, wells were washed with excess PBS, and standard media was replaced with glucose-free media. PBMC were then counted, washed in excess media, resuspended in glucose-free media and added to PC3-LN3-PSMA or MCF7 cultures. Glucose or fructose (both from Sigma) dissolved in PBS was then added to cultures to achieve the indicated concentration.
Cytotoxicity Assay
Cytotoxicity of CAR transduced cells toward PC3-LN3-PSMA or MCF7 cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After co-culture of PBMC and PC3-LN3-PSMA or MCF7 cells, the remaining media was completely aspirated and replaced with fresh DMEM supplemented with 10% FBS and 500 mg/mL MTT (Sigma). Plates were then incubated for 90 min at 37°C in 5% CO2. All media was then aspirated and formazan crystals resulting from remaining viable PC3-LN3-PSMA or MCF7 cells were solubilised in Dimethyl sulfoxide (DMSO) (Sigma). The absorbance of this solution was measured at 590nm using a FLUOstar Omega plate reader. Cytotoxicity was determined relative to control wells where PC3-LN3-PSMA or MCF7 cells had been cultured in the absence of PBMCs. For cultures containing defined concentrations of glucose or fructose, cytotoxicity was determined relative to PC3-LN3-PSMA or MCF7 cells cultured in the same concentration of glucose or fructose. The viability of target cell cultures was calculated using the formula:
Viability = Absorbance of Tumor Only x 100’ instead of ‘% Viability = (well Absorbance / Tumor only absorbance) x 100
Surface cell staining
For staining of cells with unconjugated or fluorescently conjugated primary antibodies, cells were washed twice in 1 mL of staining buffer (PBS supplemented with 5% FBS) and then incubated with 100 µL of primary antibody at the indicated dilution for 30 min at 4°C. Cells were then washed in 1 mL of staining buffer. If using primary conjugated antibodies, cells were resuspended in 300 µL of staining buffer and analysed by flow cytometry. If stained with an unconjugated primary antibody, cells were incubated with 100 µL of fluorophore-conjugated secondary antibody at the indicated dilution for 30 min at 4°C. Cells were then washed in 1 mL of staining buffer, resuspended in 300 µL of staining buffer and analysed by flow cytometry. The following antibodies were used for cell surface protein expression; mouse anti-myc (9E10, 1:4, in-house production), mouse anti-human CD3 FITC (UCHT1, 1:40, Biolegend), mouse anti-human PD-1 PE (EH12.2H7, 1:40, Biolegend), mouse anti-human CD69 APC (FN50, 1:40, Biolegend), Goat anti-mouse PR (Poly4053, 1:100, Biolegend).
All samples for flow cytometry were acquired using either a BD LSRFortessa Flow Cytometer and BD FACSDiva software or a Beckman CytoFLEX and CytExpert software. Flow cytometry data were analysed using FlowJo software (BD).
Assessment of cell viability by flow cytometry
Cellular viability was determined by staining with FITC-conjugated Annexin V (Biolegend) and Zombie Near Infared (NIR) Live/Dead (L/D) stain (Biolegend). Cells were first washed in PBS before being resuspended in 100 mL of Annexin V Binding Buffer (Biolegend) containing Annexin V (1:20 dilution) and Zombie NIR L/D (1:1000 dilution) and incubated for 30 mins at 4°C. Cells were then washed twice in Annexin V binding buffer before being resuspended in 300 mL of Annexin V staining buffer and analysed by flow cytometry.
Intracellular cytokine staining (ICS)
6 hours before intracellular cytokine staining, 5 µg/mL of brefeldin A (Biolegend) was added to PBMC cultures. Cells were washed twice in staining buffer and, if required, stained with antibodies against cell surface antigens as described above. Cells were then fixed by resuspending them in 200 µL of 4% formalin and incubated at room temperature for 20 mins. Fixed cells were then washed once in 1 mL of staining buffer before being resuspended in 100 µL of permeabilisation (perm) buffer (1% Bovine Serum Albumin, 0.1% Saponin in PBS) and incubated at 4°C for 15 mins. Intracellular antibodies were then added directly to cells to achieve the final dilution indicated and cells were incubated at 4°C for 15 mins. Finally, cells were washed twice in 1 mL of perm buffer, resuspended in 300 µL of perm buffer and analysed by flow cytometry. The following antibodies were used for ICS; mouse anti-human TNFa PE (Mab11, 1:20, Biolegend), mouse anti-human IL-2 PerCP/Cy5.5 (MQ1-17H12, 1:20, Biolegend), mouse anti-human INFg APC (4S.B3, 1:20, Biolegend).
Cell Enumeration with CountBright Beads
For counting cells with CountBright beads, cells were first washed twice in 1 mL of staining buffer. If necessary, cells were then stained for cell-surface protein expression as described above. Cells were then resuspended in 275 µL of staining buffer and 25 µL of CountBright Beads (Invitrogen) were added to each sample. Samples were then analysed by flow cytometry and the number of cells events and bead events tabulated. The cell concentration was determined using the following formula:
Cell Count cells𝜇L = Cell Count × 25Bead Count × 275×Counting Bead Concentration (beads𝜇L)
Analysis of proliferation with CellTrace Far Red
For analysing PBMC proliferation, PBMCs were counted, washed in excess PBS and resuspended to a density of 1x106 cells/mL in PBS containing 1µM CellTrace Far Red (Invitrogen). PBMC suspensions were then incubated at 37°C for 20 mins. Cells were then washed in excess RPMI supplemented with 5% human serum, resuspended in an appropriate volume of media and either cocultured with PC3-PSMA cells or stimulated with PMA and Ionomycin. 72–96 hrs after stimulation, cells were washed twice in 1 mL of staining buffer. If necessary, cells were then stained for cell-surface protein expression as described above. Cells were then resuspended in 300 µL of staining buffer and Cell Trace Far Red fluorescent signal was determined by flow cytometry.
Western Blot
Protein lysates were generated from PBMCs by first resuspending cells in 100 µL of Radioimmunoprecipitation Assay buffer (150 mM NaCl, 1% Triton-X100, 0.5% Sodium Deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) and incubating on ice for 30 mins. Lysates were then cleared by centrifugation at 16000 g for 30 mins at 4°C. Cleared lysates were transferred to 1.5 mL conical tubes and stored at -20°C until use. Total protein concentration of lysates was determined using the BCA Protein Assay Kit (Thermo Scientific), as per manufacturer instructions.
For polyacrylamide gel electrophoresis, 20 µg of protein was first mixed with 2X sample loading buffer (4% SDS, 10% 2-ME, 20% glycerol, 0.004% bromophenol blue, 125 mM Tris-HCl pH 6.8) and boiled at 95°C for 5 mins. Lysates were then separated on NuPAGE 4%-12% Bis-Tris Mini Protein Gels (Invitrogen) at 120 V for 90 mins before transfer to a PVDF membrane at 100 V for 90 mins at 4°C. Membranes were then blocked by incubating in blocking buffer (5% non-fat dried milk dissolved in PBS containing 0.1% Tween-20 (PBS-T)) for 1 hr at room temperature. Membranes were then incubated for 18 hrs in primary antibody diluted in blocking buffer at 4°C. Membranes were washed 3 times in PBS-T before being incubated with HRP-conjugated secondary antibody diluted in blocking buffer for 1 h. Membranes were then washed 3 times in PBS-T before Enhanced Chemiluminescent System (ECL) was used to image the signal with a Chemidoc XRS + imaging system (Bio-rad). The following antibodies were used for western blotting; mouse anti-human GLUT5 (E-2, 1:50, SCBT), rabbit anti-human b-actin (D6A8, 1:100, CST), goat anti-mouse HRP (1:1000, Invitrogen) goat anti-rabbit HRP (1:1000, Invitrogen).
The Cancer Genome Atlas (TCGA) Analysis
TCGA expression data and plots were obtained using GEPIA2 web server Expression DIY tool1.
Metabolite quantification
Glucose, Fructose and Lactate concentrations were quantified from sera or cell culture media using D-Glucose/D-Fructose Assay Kit or D-Lactate Kit (Megazyme).
For glucose/fructose concentration determination, 1 µL of cell culture supernatant, sera or glucose/fructose standards was dispensed into a 96 well plate. 100 µL of water, 5 µL of solution I (buffer) and 5 µL of solution II (NADP+/ATP) was added to each well, and optical absorbance was measured at 340 nm (A1). 1 µL of solution III (HK/G6P-DH) was added to each well, samples were gently mixed, incubated at room temperature for 5 mins and optical absorbance was measured again at 340 nm (A2). Finally, 1 µL of solution IV (PGI) was added to each well and mixed gently. The plate was incubated at room temperature for 10 mins before optical absorbance was measured for a final time at 340 nm (A3). Absorbance for glucose was calculated as A2-A1. Absorbance for fructose was calculated as A3-A2-A1. Absolute glucose and fructose concentrations were determined by plotting a standard curve of absorbance against the concentration of glucose/fructose standards, and interpolating the absorbance observed in each sample against this standard curve.
For lactate concentration determination, 1 µL of cell culture supernatant or lactate standards was dispensed into a 96 well plate. 100 µL of water, 5 µL of solution I (buffer), 5 µL of solution II (NADP+/PVP) and 1 µL of solution III (D-GPT) was added to each well, and optical absorbance was measured at 340 nm (A1). 1 µL of solution IV (LDH) was added to each well, samples were gently mixed, incubated at room temperature for 5 mins and optical absorbance was measured again at 340 nm (A2). Absorbance for lactate was calculated as A2-A1. Absolute lactate concentrations were determined by plotting a standard curve of absorbance against the concentration of lactate standards, and interpolating the absorbance observed in each sample against this standard curve. In all experiments absorbance was measured using a FLUOstar Omega plate reader.
Xenograft Model and adoptive CAR T-cell therapy
All animal experiments were conducted in accordance with UK Home Office guidelines under the project licence P23115EBF. NOD scid gamma (NSG) mice were maintained under specific-pathogen free conditions.
The pharmacokinetics of intraperitoneal (IP) fructose delivery was determined by injecting mice with 300 mg/kg fructose dissolved in PBS via IP injection. At specified time points post-IP injection, 20 µL blood samples were collected from mice tail veins. Serum samples were obtained by centrifuging blood samples at 2000 g for 10 mins and transferring serum to a 1.5 mL conical tube. Samples were stored at -20°C until use.
For CAR efficacy studies, mice were subcutaneously inoculated with 2.5x106 PC3-LN3-PSMA cells. 9 days after tumour engraftment, mice were intravenously injected with 1x106 CAR+ T cells. After CAR T cell adoptive transfer, mice received daily intraperitoneal injections of fructose (300 mg/kg). Tumour growth was monitored every 2–3 days by callipers measurement. Tumour volume was calculated as length × width2 × (π / 6). Mice weight was monitored every 7 days. For survival analysis, mice were humanely culled when either (1) tumour measured > 13.5mm in any direction, (2) tumour ulcerated, (3) > 15% weight loss or (4) mice exhibited poor mobility or piloerection.