Exploiting oxidative phosphorylation to promote the stem and
immunoevasive properties of pancreatic cancer stem cells

Primary human PDAC cells

PDAC PDXs were obtained from Dr. Manuel Hidalgo under a Material Transfer Agreement with the Spanish National Cancer Centre (CNIO), Madrid, Spain (Reference no. I409181220BSMH) and were originally described and genetically characterized in ref. 47. For primary cultures, PDX-derived tumor tissue fragments were minced, enzymatically digested with collagenase (Cat no. 07416, Stem Cell Technologies) for 60 min at 37 °C, and after centrifugation for 5 min at 500 × g the pellets were resuspended and cultured in RPMI, 10% fetal bovine serum (FBS), and 50 units/ml penicillin/streptomycin as described previously7. PDAC PDX-derived cultures are referred to by a random number designation [i.e., PANC185, PANC185scd (single-cell-derived)7, PANCA6L, and PANC286]. Primary cultures were tested for Mycoplasma at least every 4 weeks.

Establishment of OXPHOS-competent and -independent cultures

Low passage (<15) PDX-derived primary PDAC cultures were trypsinized and seeded at a concentration of 800,000 cells in p100 plates with RPMI medium supplemented with 10% FBS and 50 units/ml of penicillin and streptomycin. After 24 h, cells were cultured with either glucose-free Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) supplemented with 5 mM glucose (0.9 g/l), 10% FBS, 50 units/ml of penicillin and streptomycin, and 1 mM of pyruvate [Glucose: OXPHOS-independent conditions] or glucose-free DMEM medium (Thermo Fisher Scientific) supplemented with 5 mM galactose (0.9 g/L), 10% FBS, 50 units/ml of penicillin and streptomycin, and 1 mM of pyruvate [Galactose: OXPHOS-competent enriched conditions]. Sugar concentrations of 5 mM were chosen to mimic physiological sugar levels (glucose, 5 mM) and to avoid potential biological artifacts mediated by supraphysiological sugar levels. Media for both conditions were changed every day, over a period of 14 days, after which cells were collected for further processing and analysis, as described below. Earlier time points (e.g., 7 days) were also tested, but maximum levels of markers and gene expression were obtained at 14 days, and thus this time point was chosen for all experiments described herein. Gal-CC cultures can be maintained in culture indefinitely (>70 days tested), trypsinized, and passaged if necessary, as well as cryopreserved for future thawing and usage.

Flow cytometry and sorting

Cells were resuspended in Flow buffer [1× phosphate-buffered saline (PBS); 3% FBS (v/v); 3 mM EDTA (v/v)] before analysis with a 4-laser Attune NxT Acoustic Cytometer (Thermo Fisher Scientific). Cytometry data were acquired with the Invitrogen™ Attune™ NxT software, version 3.1.1, unless specified otherwise. For cell surface marker expression, refer to antibodies listed in Supplementary Table 1. For detection of PARKIN by cytometry, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min, washed, and permeabilized for 15 min with 1% Triton X-100. For Annexin-V staining, floating and attached cells were pooled and resuspended in 1× Annexin-V staining buffer containing Annexin-V-fluorescein isothiocyanate (FITC) diluted 1:20 (Cat no. 29001, Biotium, Freemont, CA) and incubated for 20 min at room temperature (RT) prior to flow cytometric analysis. For autofluorescent detection, cells were excited with blue laser 488 nm and selected as intersection with the filters 530/40 (FITC) and 580/30 (phycoerythrin (PE))7. For all assays, 2 mg/ml 4,6-diamidino-2-phenylindole (DAPI; Cat no. D9564, Sigma) was used to exclude dead cells with laser VL1. Data were analyzed with the FlowJo 9.3 software (Tree Star Inc., Ashland, OR.). For cell sorting, cells were resuspended in sorting buffer [1× PBS; 0.1% FBS (v/v); 3 mM EDTA (v/v)] and a FACS Vantage SE Flow Cytometer was used and events were analyzed by the BD FACSDivaTM software v.9.0 (BD Biosciences, San Jose, CA). Examples of gating strategies for all of the aforementioned cytometry-based analyses are presented in Supplementary Fig. 1d.

For mitochondria markers, Gluc-CC and Gal-CC were stained with Mitotracker Deep Red (Cat no. M22426), CM-H2XRos (Cat no. M7513), MitoTracker® Red CMXRos (Cat no. M7512), Mitotracker Green FM (Cat no. M7514), and NAO (Cat no. A1372) (all from Invitrogen) in the absence of FBS for 30 min at 37 °C, at concentrations of 2, 100, and 10 nM, respectively, and 0.1 µM for Mitotracker Green FM and NAO. Fluorescence was detected using the filters RL1 (allophycocyanin), YL1 (PE), and BL1 (FITC). MitoSOX (Cat no. M36008, Invitrogen) was used at 5 µM for 30 min at 37 °C and detected with laser YL1 (PE). MitoBlue (kindly provided by Dr. JL Mascareñas, Universidad de Santiago de Compostela) was used at 10 µM for 20 min at 37 °C and detected with laser VL2. Data were analyzed with the FlowJo 9.3 software (Tree Star Inc., Ashland, OR).

Cell cycle analysis

One million cells were plated in 100 mm tissue-culture dishes, and after 24 h, their cell cycles were synchronized using serum-free medium. Twenty-four hours later, treatments with glucose and galactose media were initiated and cell cycle was determined 6 days later. Cells were trypsinized and fixed in cold 70% ethanol and stored at −20 °C. After 24 h, cells were incubated with 100 µg/ml RNAase A (Sigma) in PBS during 30 min at 37 °C and then stained with 50 µg/ml of PI solution (Cat no. P4864, Sigma) and stored overnight at 4 °C. Approximately 20,000 cells/sample were analyzed using an Attune NxT Acoustic Cytometer (Thermo Fisher Scientific) with excitation at 561 nm and emission at 615 nm. The percentage of cells in each phase of the cell cycle was determined using the FlowJo 9.3 software (Tree Star Inc., Ashland, OR).

WB analysis

Cells were harvested in RIPA buffer (Cat no. R0278, Sigma) supplemented with protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Fifty micrograms of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia, Piscataway, NJ). Membranes were sequentially blocked with 1× TBS containing 5% bovine serum albumin (BSA; w/v) and 0.1% Tween20 (v/v), incubated with a 1:1000 dilution of the indicated antibodies (Supplementary Table 1) overnight at 4 °C, washed 3 times with 1× PBS containing 0.05% Tween20 (v/v), incubated with horseradish peroxidase-conjugated donkey anti-rabbit or sheep anti-mouse antibody (Amersham), and washed again to remove unbound antibody. Bound antibody complexes were detected with SuperSignal chemiluminescent substrate (Amersham) with a MyECL Imager (Thermo Fisher Scientific). Uncropped and unprocessed scans of all the main blots presented can be found in the Source data file.

Sphere-formation assay

PDAC spheres were generated and expanded in DMEM-F12 (Invitrogen) supplemented with B-27 (Cat no. A3582801, GIBCO) and basic fibroblast growth factor (PeproTech EC). One thousand cells/ml/well were seeded in ultra-low attachment 24-well plates (Corning) as described previously48. For serial passaging, spheres were harvested at day 7 using a 40-µm cell strainer, dissociated to single cells with trypsin, and then re-grown in the same conditions for 7 days (14 days total). Numbers of spheres were determined by microscopy using an inverted EVOS FL microscope (Thermo Fisher Scientific) using a ×10 objective with phase contrast.

RNA extraction and reverse transcriptase quantitative PCR (RT-qPCR)

RNA was isolated by using the standard protocol of the guanidine thiocyanate (GTC) method49. One microgram of purified RNA was used for the synthesis of cDNA using the Thermo Scientific Maxima First Strand cDNA Synthesis Kit (Cat no. K1672, Thermo Fisher Scientific) according to the manufacturer’s instructions, followed by RT-qPCR analysis using SYBR green and a StepOne Plus real-time thermo-cycler (Applied Biosystems). The thermal cycle consisted of an initial denaturation step of 10 min at 95 °C followed by 40 cycles of denaturation (15 s at 95 °C) and annealing (1 min at 60 °C). The results obtained for each gene were normalized with the β-actin levels. For primers used, see Supplementary Table 2.

Lentivirus production and cell transduction

Lentiviral particles were produced by co-transfection of 293T cells (kindly provided by Dr. Amparo Cano, Universidad Autónoma de Madrid, Spain) with the indicated plasmids using the polyethylenimine-based transfection method. Briefly, 5 × 106 293T cells were co-transfected with 1 μg packaging plasmid psPAX2, 1 μg envelope plasmid VSVG, and 2 μg of H2B-mCherry or the NANOG reporter lentiviral vector backbone (the sequence of the construct has been previously described in the publication by Hotta et al.50). After 8 h, the transfection medium was changed and recombinant lentiviruses were harvested 48 and 72 h later. The virus particle-containing supernatant was filtered through 0.45-µm PVDF membrane filters and aliquoted and stored at −80 °C until needed. For lentivirus transduction, PDAC cells were seeded in 6-well plates at a concentration of 3–5 × 104 cells/well. One milliliter of virus was directly overlaid on cells and polybrene (Cat no. 638133, Sigma) was added at a final concentration of 8 μg/ml. After 16 h, medium was changed. Stably transduced cells were obtained after mCherry-positive or YFP-positive cell sorting using a FACS Vantage SE Flow Cytometer and analyzed by the BD FACSDiva software (BD Biosciences).

Electron microscopy

After sorting cells for the indicated markers, cells were centrifuged and pellets were fixed with 0.1 M cacodylate buffer with a pH of 7.4 at RT and sections were processed by the UAM Electron Microscopy unit per standard protocols. Pictures were taken with a JEM-1010 transmission electron microscope (JEOL, USA) and analyzed by Adobe PhotoShop CS4 EXTENDED V11.0 (Adobe Systems, USA).

Optical microscopy

All the samples processed for optical microscopy were visualized and photographed with an Axiovert 135 TV microscope (ZEISS, Germany) equipped with an Olympus DP50 digital camera and a fluorescence lamp. The images were obtained with the program ViewFinderTM 7.1 (Better Light, USA) and processed with the program Adobe PhotoShop CS4 EXTENDED V11.0 (Adobe systems, USA).

Real-time proliferation assay

Fifty thousand PDAC cells were seeded in a 96-well plate. After 24 h, cells were cultured with glucose or galactose media for 14 days. Real-time analysis of proliferation and autofluorescence was performed by IncuCyte® Zoom System (ESSEN BioScience, USA) taking images every 30 min for 40 h. The results were analyzed with the IncuCyte Zoom 2015A software (ESSEN BioScience, USA).

Fluorescence microscopy

Non-sorted and sorted cells cultured with glucose or galactose were seeded in 24-well culture dishes (Corning) and incubated at 37 °C. The mitochondrial probes MitoTracker DeepRed and MitoTracker CMXRos were added at 2 and 10 nM, respectively, for 30 min at 37 °C (all from Invitrogen), followed by two washes with 1× PBS, and a final 5 min incubation with DAPI (2 mg/ml, Sigma). MitoTracker DeepRed was excited at 644 nm and the fluorescence emitted was detected at 665 nm (far red fluorescence) and assigned red fluorescence. MitoTracker CMXRos was excited at 579 nm and the fluorescence emitted was detected at 599 nm and assigned green fluorescence. For IF assays, cells were fixed with 4% PFA in PBS for 20 min at RT, washed with PBS, permeabilized with 1% Triton X-100 in PBS for 15 min, blocked with 1% BSA in PBS for 1 h at RT, and then incubated with specific antibodies (Supplementary Table 1) in a solution of 1% BSA in PBS. ProLong® Gold Antifade Reagent with DAPI (Cat no. P36941) was then added to mark cell nuclei. The fluorescent images were collected with a laser scanning confocal microscope Zeiss 710 and analyzed using the software Zen2009 5.5 (Oberkochen, Germany).

In vivo assays

For in vivo tumorigenicity assays, serial dilutions of Gluc-CC and Gal-CC resuspended in MatrigelTM (Cat no. 356234, Corning) were subcutaneously injected into 8-week-old female nude mice (Hsd:Athymic Nude-Foxn1nu/Foxn1+; Envigo) and tracked for 10–14 weeks for tumor formation. If a mouse in a specific dilution group warranted sacrifice, all of the mice (Gluc-CC and Gal-CC) in that dilution group were sacrificed in order to obtain the number of tumors for all mice at the exact same time. For the dilutions 50,000, 10,000 and 5000, mice were sacrificed at 10 weeks post inoculation. For the 1000-cell dilution group, mice were sacrificed at 14 weeks post inoculation. CSC frequencies were calculated using the ELDA software (http://bioinf.wehi.edu.au/software/elda/). For in vivo tail vein injection invasion assays, 8-week-old NOD-SCID mice (Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM) were injected intravenously via the tail vein with either 5 × 105 mCherry-H2B-labeled PANC185scd or PANCA6L Gluc-CC or Gal-CC (resuspended in 0.9% physiological saline solution) using a 27-G needle. The mice were sacrificed 10 dpi or 3 months pi. Indicated organs were collected, digested, and stained with antibodies to detect TAMs (CD45+CD11b+F4-80+) (Supplementary Table 1). In parallel, the percentage of mCherry+ cells present in each organ was determined by FACS using an Attune NxT Acoustic Cytometer (Thermo Fisher Scientific). The percentage of cells was determined using the FlowJo 9.3 software (Tree Star Inc., Ashland, OR). Organs were also analyzed by IVIS-Lumina II (Caliper Life Sciences, Hopkinton, MA, USA) and analyzed by the software Living image 3.2 (Perkin Elmer, Waltham, MA, USA).

For all in vivo experiments, mice were housed according to institutional guidelines and all experimental procedures were performed in compliance with the institutional guidelines for the welfare of experimental animals approved by the Universidad Autónoma de Madrid Ethics Committee (CEI 60-1057-A068 and CEI 103-1958-A337) and La Comunidad de Madrid (PROEX 335/14 and PROEX 294/19) and in accordance with the guidelines for Ethical Conduct in the Care and Use of Animals as stated in The International Guiding Principles for Biomedical Research involving Animals, developed by the Council for International Organizations of Medical Sciences (CIOMS). Briefly, mice were housed according to the following guidelines: a 12-h light/12-h dark cycle, with no access during the dark cycle; temperatures of 65–75 °F (~18–23 °C) with 40–60% humidity; a standard diet with fat content ranging from 4–11%; sterilized water was accessible at all times; for handling, mice were manipulated gently and as little as possible; noises, vibrations, and odors were minimized to prevent stress and decreased breeding performance; and enrichment was always used per the facility’s guidelines to help alleviate stress.

Immunohistochemistry

For histopathological analysis, 3-μm sections of formalin-fixed paraffin-embedded blocks were used for IHC analysis of human cytokeratin 19 using the primary and secondary antibodies and dilutions detailed in Supplementary Table 1. Antigens were visualized using 3,3-diaminobenzidine tetrahydrochloride plus (DAB+). Counterstaining was performed with hematoxylin. Digital images were obtained using an Axiophot (Carl Zeiss) microscope with a ×10 objective and DP70 Olympus camera. Images were processed using the ImageJ v2.0.0 software (NIH, USA, http://rsbweb.nih.gov/ij/).

Autophagy flux assay

For autophagy flux analysis, Gluc-CC and Gal-CC were treated with the lysosomotropic reagent Bafilomycin A1 at a concentration of 150 nM (Calbiochem, Thermo Fisher Scientific, Waltham, MA, USA) for 5 h. After treatment, Gluc-CC and Gal-CC were harvested and analyzed by WB as described above. For densitometry, WB images were analyzed using PhotoShop CS4 EXTENDED V11.0 (Adobe Systems, USA). Autophagy flux compares the LC3B-II (Sigma Aldrich, St. Louis, MO, USA) levels with and without the autophagy inhibitor and is calculated as the ratio of LC3B-II in the presence and absence of the autophagy inhibitor compared to the control condition (Gal-CC versus Gluc-CC). The data were normalized to the indicated housekeeping protein.

Quiescence assay

Gluc-CC and Gal-CC were labeled with PKH26 red fluorescent cell membrane labeling dye (Cat no. MINI26-1KT, Sigma) according to the manufacturer’s instructions. At the indicated days post staining, for a total of 14 days, cells were trypsinized, washed, and the percentage of PKH26+ cells was determined using an Attune NxT Acoustic Cytometer (Thermo Fisher Scientific). DAPI (Sigma) was used to exclude dead cells with laser VL1.

Senescence assay

β-Galactosidase activity was measured in Gluc-CC y Gal-CC according to the manufacturer’s instructions (Cat no. 94433, Sigma). β-Galactosidase+ cells were determined using an Axiovert 135 TV optical microscope (ZEISS, Germany), and the ImageJ 2.0.0 software (NIH, USA) was used for further analysis.

Chemoresistance in vitro assays

PDAC cells were seeded in a 12-well plate at 106 cells/well. Following 14 days of culture with glucose or galactose, Gal-CC or Gluc-CC cells were treated with 1 μg/ml of GEM, 3 µM of MTX, or 1 µM of DXR for 2 days. After 2 days, drugs were removed and PDAC cells were allowed to recover for 3 days. For metabolic drugs, Gluc-CC and Gal-CC were treated with 5 µM of Menadione, 10 nM of Rotenone, 100 µM of Resveratrol, 3 mM of Metformin, or 5 mM of 2-DG for 3 days. Following treatments, the supernatant was collected, and the ToxiLight™ Bioassay Kit (Cat no. LT07-217, Lonza, Switzerland) was used to evaluate the toxicity using a bioluminescence GLOMAX Luminometer (Promega).

Measurement of mitochondrial metabolism by Seahorse analyzer

Measurements of OCR were performed with a XF extracellular flux analyzer (Seahorse Bioscience, Agilent Technologies, Santa Clara, CA, USA), and data were collected with the XF96 1.4.2 Software (Agilent). Briefly, 14-day-old Gal-CC or Gluc-CC were trypsinized and re-seeded at a density of 105 cells per well in a XF24 cell culture microplate with DMEM (4.5 g/L glucose, +L-Glutamine, -Pyruvate, pH 7.6 at RT) supplemented with 10% heat-inactivated FBS (Gibco) and 1% Pen/Strep. On the day of the assay (24 h after re-seeding), medium was changed to non-carbonated Seahorse measurement medium and the XF24 plate was transferred to a temperature-controlled (37 °C) XF24 Extracellular Flux analyzer and equilibrated for 10 min. To determine the basal respiration, four assay cycles (1-min mix, 2-min wait, and 3-min measuring period) were performed. Then oligomycin (4 μM) was added by automatic pneumatic injection (three assay cycles) to inhibit ATP synthase and thus approximate the proportion of respiration used to drive ATP synthesis versus proton leak-linked respiration. Oligomycin was followed by an injection of FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) (0.5 μM) to completely dissipate proton motive force and maximally stimulate mitochondrial respiration (three assay cycles), thus determining SRC and substrate oxidation capacity. An injection of rotenone (4 μM) and antimycin A (2 μM) was used to correct for the non-mitochondrial respiration rate (three assay cycles), which was subtracted from all the other rates. Basal respiration was calculated as the oligomycin-sensitive fraction of mitochondrial respiratory activity, estimating the proportion of basal respiration used to drive ATP synthesis. To determine ECARs, experiments were terminated by injection of 2-DG (100 mM), correcting for non-glycolytic acidification. Injection of 2-DG enables the calculation of glycolytic acidification. Data obtained after oligomycin treatment induced glycolytic capacity. Raw data were normalized to total cell numbers, determined by optical absorbance of lysed crystal violet-stained cells.

RNA sequencing analysis

Total RNA was isolated by the GTC method using standard protocols49. Sequencing libraries were prepared with the “NEBNext Ultra II Directional RNA Library Prep Kit for Illumina” (NEB no. E7760) as recommended by the kit manufacturer. Briefly, polyA+ fraction was purified and randomly fragmented, converted to double-stranded cDNA, and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters. Adapter-ligated library was completed by PCR with Illumina PE primers. The resulting purified cDNA libraries were applied to an Illumina flow cell for cluster generation and sequenced on an Illumina NextSeq 550 (with v2.5 reagent kits), according to the manufacturer’s protocols. RNAseq data sets were analyzed using the tool Nextpresso51. Nextpresso is comprised of four basic levels: (1) quality check, (2) read cleaning and/or down-sampling, (3) alignment, and (4) analysis (gene/isoform expression quantification, differential expression, gene set enrichment analysis, and fusion prediction). The gene signatures (Hallmark genesets, h.all.v7.1.symbold.gmt) from GSEA—Molecular Signature Database for Gene set enrichment analysis was used for pathway enrichment analysis with GSEA v4.0.3 (Broad Institute).

Zebrafish maintenance and xenograft assays

Zebrafish embryos were obtained by mating adult zebrafish (Danio rerio, wild type), maintained in 30-l tanks with a ratio of 1 fish per liter of water, with 14 h/10 h light/dark cycle and a temperature of 28.5 °C according to published procedures35. All the procedures used in the experiments as well as fish care were performed in agreement with the Animal Care and Use Committee of the University of Santiago de Compostela and the standard protocols of Spain (Directive 2012-63-UE). At the final point of the experiments, zebrafish embryos were euthanized by tricaine overdose.

For zebrafish xenograft assays and image analysis, zebrafish embryos were collected at 0 h post-fertilization (hpf) and incubated until 48 hpf at 28.5 °C. At 48 hpf, hatched embryos were anesthetized with 0.003% of tricaine (Sigma). mCherry-H2B-labeled PANC185scd and PANCA6L Gluc-CC and Gal-CC were trypsinized, resuspended, and concentrated in an Eppendorf at 106 cells per tube for each condition. Cells were then resuspended in 10 µl of PBS with 2% polyvinylpyrrolidone to avoid cellular aggregation. Borosilicate needles (1 mm O.D. × 0.75 mm I.D.; World Precision Instruments) were used to perform the xenograft assays in the zebrafish embryos. Between 100 and 150 cells were injected into the circulation of each fish (Duct of Cuvier) using a microinjector (IM-31 Electric Microinjector, Narishige) with an output pressure of 34 kPA and 30 ms of injection time per injection. Subsequently, the injected embryos were incubated at a temperature of 34 °C for 6 dpi in 30 ml Petri dishes for each condition with salt dechlorinate tap water. Imaging of the injected embryos was performed using a fluorescence stereomicroscope (AZ-100, Nikon) at 1, 4, and 6 dpi in order to measure the proliferation of the PANC185scd- and PANCA6L-injected human cancer cells inside the zebrafish circulation in each of the conditions assayed.

The image analysis of the injected embryos was carried out using the Quantifish software v2.1 (University College London, London, UK) in order to obtain the proliferation ratio of the cells in the region of the caudal hematopoietic tissue of the embryos, where the cells proliferate and metastasize. This program measures in each of the images provided the intensity of the fluorescence and the area of the positive pixel above a certain threshold of the cells. With these parameters, an integrated density value is obtained allowing the researcher to compare different times between images to reach a proliferation ratio.

Lactate production assay

Supernatant from Glu-CC and Gal-CC were collected to evaluate the changes in the levels of lactate production. The analysis was performed using the Lactate Assay Kit (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The optical density was determined using a Synergy™ HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) at a wavelength set to 570 nm.

ATP determination assay

Lysate pellets of cells from Glu-CC and Gal-CC were collected to evaluate the changes in the levels of ATP. The analysis was performed using the Molecular Probes ATP Determination Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Bioluminescence was determined using a Synergy™ HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA).

Isolation of macrophages and T cells from human blood

Blood samples from healthy donors were provided by the BioBank Hospital Ramón y Cajal-IRYCIS (PT13/0010/0002), integrated in the Spanish National Biobanks Network (ISCIII Biobank Register No.  B.0000678). Samples were processed following standard operating procedures with the appropriate approval of the Hospital Ramón y Cajal Ethical and Scientific Committee (Control No.: DE-BIOB-73 AC65, RG.BIOB-57, and RG.BIOB-54), with informed consent and according to Declaration of Helsinki principles. Blood samples were diluted with PBS (Cat no. 10010023, Gibco) and Ficoll (Cat no. L6115, Merck) was used to isolate peripheral blood mononuclear cells (PBMCs). PBMCs were divided across three 6-well culture plates (per donor) with RPMI 1640 media containing 10% FBS and 50 units/ml penicillin/streptomycin. After 24 h, monocytes adhered to the plate surface were separated from the lymphocytes that remain in suspension and that were seeded in T-75 flasks for their subsequent activation and growth.

Cytokine array

Changes in protein levels related to immune evasion processes were determined by measuring changes in the levels of cytokines or inflammatory-related molecules released into the culture medium by Gluc-CC and Gal-CC cells, using the Proteome Profiler Human Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Cytokine array images were obtained using the MyECL Imager (Thermo Fisher Scientific). For densitometry analysis, array images were analyzed using PhotoShop CS4 EXTENDED V11.0 (Adobe Systems, USA). Data were normalized to the indicated array positive control.

TFGβ secretion assay

Culture medium from Gluc-CC and Gal-CC were collected to evaluate the changes in the levels of TGFβ secretion. The analysis was performed using the Human TGFβ1 Immunoassay Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The optical density was determined using a Synergy™ HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) at a wavelength set to 450 nm.

Macrophage immunoevasion assay

Five days after isolation of macrophages from the blood of healthy donors (see above), macrophages were labeled with the red membrane dye DilC, according to the manufacturer’s instructions (Cat no. D3911, Thermo Fisher). Gluc-CC and Gal-CC were harvested and labeled with PKH67 green fluorescent cell membrane labeling dye (Sigma) according to the manufacturer’s instructions. Labeled cells were seeded together with labeled macrophages during 24 h. Afterwards, macrophage-mediated phagocytosis (DilC+/PKH67+ cell percentages) was determined using the AttuneNxT cytometer (Thermo Fisher Scientific), and data were analyzed with the FlowJo 9.3 software (TreeStar, Ashland, OR).

T cell immunoevasion assay

Seven days after isolation and expansion of T cells from the blood of healthy donors, T cells were activated with phytohemagglutinin-P (Cat no. L8754, Merck) at a concentration of 5 μg/ml and with 20 ng/ml of human rIL-2 (Cat no. PHC0026, Gibco) for 7 days. Gluc-CC and Gal-CC were harvested and co-seeded with activated T cells during 10 days. Afterwards, T cells were removed and the extent of cytotoxicity was determined with the ToxiLight™ Bioassay Kit (Cat no. LT07-217, Lonza, Switzerland) using a bioluminescence GLOMAX Luminometer (Promega). The data were analyzed with the Prism 8 software (San Diego, CA).

Statistical analysis and reproducibility

Results are presented as means ± standard deviation (stdev) or ±standard error of the mean (SEM), as indicated in the figure legends. Different two-sided statistical analyses were performed: one-way analysis of variance (ANOVA; two sided) with Bonferroni adjustment or with an unpaired two-tailed t test to determine significance, two-way ANOVA with Tukey’s multiple comparisons test, or unpaired two-sided (confidence interval of 95%) t test for multiple comparisons using the Holm–Sidak method were used to determine differences between means of groups. p Values < 0.05 were considered statistically significant. GraphPad software (GraphPad Prism 8.0, GraphPad Software, La Jolla, CA, USA) was used to perform the statistical analysis of the data.

The number of biologically independent samples are indicated in the figure legends. Repeated independent experiments per each panel with similar results are shown below. n = 1 (Figs. 1a, e, 2b, 3d, l, 4a, k, 5c, k, 6e, n, 7g, k and Supplementary Figs. 1a, c, h, i, 2a–e, h–j, m, 4a, b, 3e, 5a, d–f, 6a, b, 7a–d); n = 2 (Figs. 1f, g, 2e–j, l, m, 3e, 5a, 6c, d, 7f, j and Supplementary Figs. 2a–e, g, 3b, d, 5b, h–k); n = 3 (Figs. 2f, 6b, 7b, d and Supplementary Figs. 1f, 3i, 6d–f), n = 4 (Figs. 2d, 3b and Supplementary Figs. 1b, 2f).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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