Lipid droplet dependent fatty acid metabolism controls the phenotype of tumor-associated macrophages (2019)
Figure 6

Figure 6. Oleate induces polarization of human monocytes into CD206+ suppressive cells.

(A) 1×106 CD14+ myeloid cells from healthy donors were polarized by 1 ng/ml GM-CSF in the presence of BSA-control (C), 0.2 mM oleate (O) or 0.2 mM stearate (S) over a 6-day period. The expression of CD206, CD204 and CD38 was analyzed via flow cytometry. (B) The level of lipid droplets was determined via BODIPY staining. (C) The oxygen consumption rate (OCR) of differentiated myeloid cells was monitored as described in Materials and Methods. (D) The purified CFSE-labeled autologous CD4+ T cells were co-cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay. (E) Tumor tissue (tumor) as well as corresponding non-tumorous adjacent tissue (control) were collected from colorectal cancer patients and prepared for histopathology. The expression of CD68 (red), CD206 (green) and ADRP (blue) indicate the lipid droplets in tumor infiltrating myeloid cells. Nuclei (white) were stained using DAPI. The absolute number of positive cells was quantified in 5 high-power fields (hpf) (scale bar = 20 µm). Shown is the mean±SD from two to four independent experiments and analyzed by either One-way ANOVA (A), Kruskal-Wallis test with Dunn's multiple comparisons test or unpaired Student's two-tailed t tests (E). *p<0.05; **p≤0.01; ***p≤0.001

Material and Methods

Cell lines. The murine colon carcinoma CT26 cells and the fibrosarcoma cell line MCA 205 were kindly provided by Prof. Zhihai Qin. The myeloid suppressor cell line MSC 2 cell line was a kind gift from Professor Ghiringhelli (Institut National de la Santé et de la Recherche Médicale (INSERM) U866, Dijon, France). All cell lines were tested for mycoplasma contamination on a monthly basis using Venor®GeM Advance Kit (Minerva Biolabs GmbH, Berlin, Germany) according to the manufacturer's protocols.

Mouse Tumor Models. All animal protocols were approved by the regional animal study committee of Berlin (Germany) and the Institutional Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences. C57BL/6 and BALB/c mice (6 - 8 weeks of age, female) were purchased from either Janvier labs or the Weitong Lihua Company and were housed under standard conditions with free access to water and autoclaved standard chow. In co-injection tumor models, 5×105 CT26 cells were mixed with indicated amount of purified myeloid cells in matrigel (BD Bioscience), and then injected subcutaneously. To test the anti-tumor effect of diacylglycerol acyltransferase (DGAT) inhibitor in vivo, intra-tumoral injection of DGAT1 inhibitor A922500 (3 mg/kg) and DGAT2 inhibitor PF-06424439 (10 mg/kg) (both Sigma-Aldrich Chemie GmbH) were administered once daily at day 6 after 5×105 CT26 tumor cell inoculation. Tumor growth and body weight was monitored daily. To test the effect of lipid droplet-formation inhibitors, 2.5×105 MCA205 cells were inoculated subcutaneously. Intraperitoneal injection of liposome-encapsulated inhibitors or liposome vehicle as control started at day 7. Tumor growth was monitored every 2-5 days and tumor volume was calculated as (length×width×width/2).

Fatty Acid-Albumin Preparation. Fatty acid salts were dissolved in hot distilled water and added rapidly to proper cell culture medium with fatty acid-free bovine serum albumin (BSA) at a molar ratio of 8:1 (fatty acid salt: albumin). Fatty acid-albumin complex solutions were freshly prepared prior to each experiment.

Affymetrix Microarray Analysis. Purified Gr1- myeloid cells were treated as indicated before 11 and total RNA was extracted by using the guanidinium isothiocyanate method (TRIzol reagent) followed by purification using the RNeasy Kit (Qiagen). RNA quality was assessed by using the Agilent Model 2100 Bioanalyzer (Agilent Technologies). Ten µg of total RNA were processed for analysis on the microarray by using the Affymetrix GeneChip one-cycle target labeling kit (Affymetrix) according to the manufacturer's protocols. The resultant biotinylated cDNA was fragmented and hybridized to the GeneChip Mouse Gene 2.0 ST Array. The arrays were washed, stained, and scanned using the Affymetrix Model 450 Fluidics Station and Affymetrix Model 3000 scanner according to the manufacturer's protocols. Expression values were generated by using Microarray Suite (MAS) v5.0 software (Affymetrix). Each sample and hybridization underwent a quality control evaluation, including cDNA amplification of >4-fold, percentage of probe sets reliably detecting between 40% and 60% present call, and 3'-5' ratio of GAPDH gene <3.

Evaluation and Normalization of Affymetrix GeneChip Data. The hybridizations were normalized by using the robust multichip averaging (RMA) method to obtain summary expression values for each probe set. Gene expression levels were analyzed on a logarithmic scale. For the assessment of the parental strains, regression, analysis of variance (ANOVA), t tests, and the Mann-Whitney-Wilcoxon rank test were used to identify differentially expressed genes. Heat maps for gene expression data were generated by web tool Clustvis (Metsalu & Vilo, 2015).

Inhibition of Lipid Droplet Formation. The combination of DGAT inhibitors (iDGAT) (DGAT 1 inhibitor: A922500; DGAT2 inhibitor: PF-06424439, adipose triglyceride lipase (ATGL) inhibitor atglistatin (SML1075, Sigma-Aldrich Chemie GmbH) as well as monoacylglycerol lipase (MAGL) inhibitor MJN110 (SML0872, Sigma-Aldrich) served to block the lipid droplets formation. Bone marrow derived myeloid cells were cultured with the indicated free fatty acid (FFA) in the presence of variant inhibitors for 7 days.

Liposome Preparation. Liposomes were prepared by extruding a suspension of dissolved hydrated lipids through Whatman Nucleopore polycarbonate membranes in a Lipex Biomembranes Extruder (Northern Lipids, Vancouver, Canada). A lipid composition of 59.87% dipalmitoylphosphatidylcholine (DPPC), 40% cholesterol, and 0.12% DSPE-PEG-peptide conjugate was used. The lipids were dissolved in ethanol at 60 °C and then hydrated for 2 h with a 400 mM ammonium sulfate solution with ethanol ≤10% of final volume, and 25 mM final lipid concentration. The suspension was passed four times with a 0.2 µm membrane and eight times with a 0.1 µm membrane through the extruder at 60 °C and a pressure of approximately 100 psi. Liposomes were dialyzed (100 kDa MWCO) against phosphate-buffered saline (PBS) to establish an ammonium sulfate gradient. Liposome size was determined by a laser particle size analyzer (zetasizer Nano) at a wavelength of 633 nm with a constant angle of 173 °.

Preparation of Peptide Conjugates. Here we synthesized a cell type specific binding of peptides (GGP peptide) for neutrophils and monocytes as conjugator for liposomes 34. Briefly, 10.9 mg (5.6 µmol) of GGP peptide were dissolved in 500 µl of dimethyl sulfoxide (DMSO). DSPE-PEG (2000)-COOH (10 mg, 3.5 µmol), dicyclohexylcarbodiimide (DCC) (10.8 mg, 53.6 µmol) and 250 µL of pyridine were added to the reaction and incubated for 6 h at ambient temperature. Pyridine was removed by rotary evaporation. Conjugate micelles were formed by hydrating the DMSO solution with 5 ml of deioinzed water until DMSO reached 10 % of the final volume. Unconjugated compounds were removed by dialysis (100 kDa MWCO) against 1 l, 50 mM NaCl (2×) and 1 l of deionized water (2×) followed by lyophilization. Final peptide content was determined using the DC protein assay (Bio-Rad, Hercules, CA, USA). The final conjugate was characterized by thin layer chromatography (TLC) on pre-coated plates (silica gel 60 F254) and MALDI-TOF mass spectroscopy (Applied Biosystems 4700 proteomics analyzer) to confirm molecular mass, with spectrum obtained in negative ion mode in a-cyano-4-hydroxycinnamic acid (CHCA) matrix.

Loading of Inhibitors into Liposomes. Liposomes were loaded with inhibitors by an ammonium sulfate gradient. Ten mg/ml A92250 (DGAT1 inhibitor) and PF-06424439 (DGAT 2 inhibitor) were encapsulated together into liposome at a ratio of 0.1 mg (A922500)/0.5 mg (PF-06424439) inhibitor per mg of DPPC in the liposomes. Etomoxir and CAY10566 were loaded by the same procedure in the ratio of 0.1 mg (CAY10566) / 0.5 mg (etomoxir) per mg of DPPC. Liposome were cooled immediately on ice and dialyzed (100 kDa MWCO) twice against PBS to remove free inhibitors. The final inhibitors concentration after dialysis was determined after liposome lysis with 10% Triton X‑100 by UV absorbance at 480 nm.

Mitochondrial Respiration Analysis. Purified polarized Gr1- murine myeloid cells or human monocytes were seeded in XF96 cell culture microplates (Agilent) in at 1×105 cells/well in 180 μl pre-warmed assay medium. To allow media temperature and pH to reach equilibrium before the first rate measurement, cells were incubated at 37 °C in 5% CO2 for 2 h. Prior to each rate measurement, the XFe96 Analyzer (Seahorse Bioscience) gently mixed the assay media in each well for 3 min to allow the oxygen partial pressure to reach equilibrium. Following mixing, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured simultaneously for 3-5 min to establish a baseline rate. The assay medium was then gently mixed again for 3-5 min between each rate measurement to restore normal oxygen tension and pH in the microenvironment surrounding the cells. After the baseline measurement, 20-27 μl of a testing agent prepared in assay medium was then injected into each well to reach the desired final working concentration. This was followed by mixing for 5-10 min to expedite compound exposure to cellular proteins, after which OCR and ECAR measurements were conducted. Generally, two to three baseline rates and two or more response rates (after compound addition) were measured, and the average of two baseline rates or test rates served for data analysis. Calculations were performed as follows: Basal Respiration = (Last rate measurement before first injection) - (Non-Mitochondrial Respiration), Spare Respiratory Capacity = (Maximal Respiration) - (Basal Respiration), Proton Leak = (Minimum rate measurement after oligomycin injection) - (Non-Mitochondrial Respiration), ATP Production = (Last rate measurement before oligomycin injection) - (Minimum rate measurement after oligomycin injection), Maximal Respiration = (Maximum rate measurement after FCCP injection) - (Non-Mitochondrial Respiration), Non-mitochondrial Respiration = Minimum rate measurement after rotenone/antimycin A injection.

Tumor Infiltrating Immune Cell. Briefly, tumor tissues were bathed in 70% isopropanol for 30 s and then transferred to a Petri dish. Tumors were minced into pieces <3 mm in diameter and digested in 2 mg/ml collagenase type D at 37 °C for 1 h. The digested tissue pieces were pressed through a 100 µm Falcon® nylon cell strainer (Corning, NY, USA). Purified cell suspensions were analyzed by flow cytometry.

Bone Marrow-derived Myeloid Cells. Bone marrow cells were isolated as previously described (Wu et al, 2017). Briefly, the cavities of femur and tibia bones of BALB/c mice were flushed with phosphate-buffered saline (PBS). The single cell suspensions were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM, 4.5 g/l D-glucose) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 40 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF, Peprotech 315-03). After 24 h of incubation, non-adherent macrophage progenitor cells were isolated and cultured in the presence of 0.2 mM BSA/sodium stearate or 0.2 mM BSA/sodium oleate plus 40 ng/ml GM-CSF for 7 days in 6-well plates. Medium was renewed every other day.

Murine T Cell Proliferation Assay. For the T cell proliferation assay, syngeneic CD4+ /CD8+ T cells were isolated from spleens and lymph nodes of healthy mice and purified via MACS (Miltenyi Biotec). The purity of CD4+ and CD8+ T cell-sorting was >98%. T cells were stained with 0.5 µM CFSE for 10 min at 37 ºC followed by thorough washing and resuspension in T cell medium. Five µg/ml concanavalin A or 96-well plates pre-coated with 5 µg/ml anti-CD3 (clone 2C11) plus 5 µg/ml soluble anti-CD28 (clone 37.51E1; both BD Pharmingen) served for T cell stimulation as indicated. Stimulated carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells were cultured in the presence or absence of beads-purified Gr1- bone marrow-derived myeloid cells in various ratios as indicated. After 72 h, the cultured cells were washed and gated on lymphocytes. The CFSE dilution due to cell division was analyzed by a FACSCanto II device (BD Biosciences) using FlowJo software (Tree Star, Inc).

Nitric Oxide Measurements. The supernatant from T cell proliferation assay was collected for nitric oxide measurements. NO production was defined as nitrite concentration using the Griess assay following manufacturer's instructions (Promega).

Arginase Activity Detection. Arginase activity was determined by measuring the amount of urea generated from the hydrolysis of L-arginine, as described previously (Zhao et al, 2012). Briefly, isolated cells were lysed in the presence of 100 μl lysis solution (0.1% Triton X-100, 10 mM MnCl2, 25 mM Tris-HCl). The lysate of Gr1+CD11b+ cells were incubated with L-arginine and the hydrolysis was stopped by adding 1 M sulfuric acid. Subsequently, α-isonitrosopropiophenone was added followed by boiling for 30 min. The concentration of urea was determined at 540 nm absorbance by the Infinite® F50/Robotic ELISA plate reader (TECAN, Männedorf, Switzerland). One U enzyme activity was defined as the amount of enzyme that catalyzes the formation of 1 μmol of urea per min.

Human Monocytes Polarization and T Cell Proliferation Assay. Peripheral blood samples were obtained from healthy volunteers, from which informed consent was obtained according to the local ethics committee of the Charité - Universitätsmedizin Berlin. All experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. PBMCs were freshly isolated by density gradient centrifugation (Bicoll; Biochrom). CD14+ cells were magnetically labeled with CD14 MicroBeads (Miltenyi Biotec) and enriched with LS columns (Miltenyi Biotec). Isolated CD14+ monocytes were taken into culture with RPMI-1640 (GIBCO, Life Technologies) supplemented with 10% fetal bovine serum (Invitrogen), penicillin/streptomycin (100 U/ml/ 100 µg/ml, Sigma-Aldrich Chemie GmbH) and with fatty acid-free BSA at a molar ratio of 8:1 (fatty acid salt: albumin) or additionally supplemented with 0.2 mM sodium oleate over 6 days. One ng/mL GM-CSF was added. After 6 days the CD14+ monocytes were co-cultured with 3×105 purified autologous CD4+ T cells for the T cell proliferation analysis. Therefore, PBMCs were isolated from the same donor and CD4+ cells were magnetically labeled with CD4 MicroBeads (Miltenyi Biotec) and enriched with LS columns (Miltenyi Biotec). The purity of CD14 and CD4 sorting was >98%. T cells were stained with 0.5 µM CFSE for 5 min at 37 °C followed by washing and resuspension in T cell medium. CFSE-labeled T cells were cultured in a 96-well plates pre-coated with 1 µg/mL anti-CD3 (OKT3; BioLegend) and 1 µg/mL anti-CD28 (CD28.2; Invitrogen) together with the CD14+ monocytes for 72 h in the ratio of 3:1. The flow cytometric analysis of the CFSE dilution due to cell division was performed by a FACSCanto II device (BD Biosciences) using FlowJo software (Tree Star, Inc).

Flow Cytometry. Flow cytometric analyses were performed using standard procedures. In brief, 2×105-108 cells were harvested and resuspended in 50 µl PBS/0.5% BSA buffer in the presence of the indicated antibodies for 10 min on ice. For boron-dipyrromethene (BODIPY) staining, cells were labeled by 0.2 µg/ml BODIPY®493/503 (Life Technologies) and incubated in 37 ºC for 15 min and washed with PBS. Data acquisition was performed using a FACSCanto II device (BD Biosciences) using FlowJo software (Tree Star).

Western Blot Analysis. For detection of mTOR, phospho-mTOR (Ser2448) and phospho-mTOR (Ser2481) (both Cell Signaling Technology), polarized myeloid cells were lysed in radioimmunoprecipitation assay buffer (RIPA buffer, Life Technologies, Carlsbad, CA, USA). Western blot was performed via standard procedure (41). Densitometric analysis was performed with the Fuji MultiGauge software (Fujifilm).

Histopathology. All human samples from colorectal cancer patients were kindly provided by the Central Biomaterial Bank Charité. Paraffin blocks of archived tissue samples were cut, dewaxed and subjected to a heat-induced epitope retrieval step. Endogenous peroxidase was blocked by hydrogen peroxide prior to incubation with anti-CD68 (clone PGM-1, Dako) followed by the EnVision+ System-HRP Labelled Polymer Anti-Mouse (Dako). For visualization, OPAL-570 diluted in amplifier diluent (PerkinElmer) was used. Proteins were then inactivated and sections incubated with anti-CD206 (clone 5C11, LifeSpanBiosciences) followed by the EnVision+ System-HRP Labelled Polymer Anti-Mouse (Dako) and OPAL-520 diluted in amplifier diluent (PerkinElmer). After inactivation of proteins, sections were incubated with polyclonal anti-ADRP (#15294-1-AP, Proteintech) followed by the EnVision+ System-HRP Labeled Polymer Anti-Rabbit (Dako) and OPAL-670 in amplifier diluent (PerkinElmer). Nuclei were stained using 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) and slides were coverslipped in Fluoromount G (Southern Biotech). Multispectral images were acquired using a Vectra® 3 imaging system (Perkin Elmer). Positive cells were quantified in 5 high power fields (field of vision in x400 original magnification). For CD8+ T cell detection, paraffin-embedded mouse tumor tissues were cut and dewaxed followed by heat-induced antigen retrieval. Sections were incubated with anti-CD8α (clone D4W2Z, CST, Germany) and PPD-540 digital image system (PANOVUE, Canada) was used following HRP-Labelled Polymer Anti-Rabbit (PANOVUE, Canada) conjugation for visualization. Nuclei were stained by using Fluoro-Gel II with DAPI (Electron Microscopy Sciences, PA, USA). All evaluations were performed in a blinded manner.

Antibodies. All antibodies used in this study, including their working dilutions, are listed in table S2.

Statistical Analysis. Statistical significance of differences between the experimental groups were determined by Student's t test or factorial analysis of variance and the respective post hoc tests (Tukey's and Dunnett's multiple comparisons test) using GraphPad Prism software (GraphPad Software, La Jolla, CA). Exact p-values for all experiments can be found in a supplementary data sheet (table S3).