ERX1649941 E-MTAB-5032:KO1 GLUT1-high_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290273 SAMEA4378824 KO1 GLUT1-high_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: Ddit4 knockout genotype Experimental Factor: high GLUT1 expression phenotype ERX1649942 E-MTAB-5032:KO1 GLUT1-low_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290274 SAMEA4378825 KO1 GLUT1-low_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: Ddit4 knockout genotype Experimental Factor: low GLUT1 expression phenotype ERX1649943 E-MTAB-5032:KO2 GLUT1-high_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290275 SAMEA4378826 KO2 GLUT1-high_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: Ddit4 knockout genotype Experimental Factor: high GLUT1 expression phenotype ERX1649944 E-MTAB-5032:KO2 GLUT1-low_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290276 SAMEA4378827 KO2 GLUT1-low_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: Ddit4 knockout genotype Experimental Factor: low GLUT1 expression phenotype ERX1649945 E-MTAB-5032:KO3 GLUT1-high_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290277 SAMEA4378828 KO3 GLUT1-high_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: Ddit4 knockout genotype Experimental Factor: high GLUT1 expression phenotype ERX1649946 E-MTAB-5032:KO3 GLUT1-low_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290278 SAMEA4378829 KO3 GLUT1-low_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: Ddit4 knockout genotype Experimental Factor: low GLUT1 expression phenotype ERX1649947 E-MTAB-5032:KO4 GLUT1-high_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290279 SAMEA4378830 KO4 GLUT1-high_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: Ddit4 knockout genotype Experimental Factor: high GLUT1 expression phenotype ERX1649948 E-MTAB-5032:KO4 GLUT1-low_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290280 SAMEA4378831 KO4 GLUT1-low_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: Ddit4 knockout genotype Experimental Factor: low GLUT1 expression phenotype ERX1649949 E-MTAB-5032:WT1 GLUT1-high_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290281 SAMEA4378832 WT1 GLUT1-high_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: wild type genotype genotype Experimental Factor: high GLUT1 expression phenotype ERX1649950 E-MTAB-5032:WT1 GLUT1-low_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290282 SAMEA4378833 WT1 GLUT1-low_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: wild type genotype genotype Experimental Factor: low GLUT1 expression phenotype ERX1649951 E-MTAB-5032:WT2 GLUT1-high_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290283 SAMEA4378834 WT2 GLUT1-high_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: wild type genotype genotype Experimental Factor: high GLUT1 expression phenotype ERX1649952 E-MTAB-5032:WT2 GLUT1-low_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290284 SAMEA4378835 WT2 GLUT1-low_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: wild type genotype genotype Experimental Factor: low GLUT1 expression phenotype ERX1649953 E-MTAB-5032:WT3 GLUT1-high_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290285 SAMEA4378836 WT3 GLUT1-high_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: wild type genotype genotype Experimental Factor: high GLUT1 expression phenotype ERX1649954 E-MTAB-5032:WT3 GLUT1-low_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290286 SAMEA4378837 WT3 GLUT1-low_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: wild type genotype genotype Experimental Factor: low GLUT1 expression phenotype ERX1649955 E-MTAB-5032:WT4 GLUT1-high_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290287 SAMEA4378838 WT4 GLUT1-high_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: wild type genotype genotype Experimental Factor: high GLUT1 expression phenotype ERX1649956 E-MTAB-5032:WT4 GLUT1-low_s ERP016834 RNA-seq analysis of normoxic and hypoxic tumor-associated macrophages ERS1290288 SAMEA4378839 WT4 GLUT1-low_s RNA-Seq TRANSCRIPTOMIC RANDOM LLC tumor-bearing mice were sacrificed by cervical dislocation and tumors were harvested. Tumors were minced in RPMI medium containing 0.1% collagenase type I and 0.2% dispase type I and incubated in the same solution for 30 minutes at 37°C. The digested tissue was filtered using a 70 μm pore sized mesh and cells were centrifuged 5 min at 1000 rpm. Red blood cell lysis was performed by using Hybri-MaxTM (Sigma-Aldrich). For flow sorting, the myeloid cell population in the tumor single cell suspension was enriched by coating with CD11b-conjugated magnetic beads (MACS, Miltenyi Biotec) and separation through magnetic columns (MACS, Miltenyi Biotec). Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and incubated for 15 minutes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 20 minutes at 4 °C: anti-F4/80 and anti-GLUT1. Cells were subsequently washed and resuspended in cold FACS buffer before flow sorting by a FACS Aria (BD Biosciences). REDD1 KO mice were generated by Lexicon Corp (USA) exclusively for Quark Pharmaceuticals Inc and are the property of Quark Pharmaceuticals Inc. WT mice were littermate controls. 5-6 weeks old C57BL/6N recipient mice were irradiated with 9.5 Gy. Subsequently, 107 bone marrow cells from the appropriate genotype were injected intravenously via the tail vein. Tumor experiments were initiated 5 weeks after bone marrow reconstitution. : 1 x 106 Lewis lung carcinoma (LLC) adherent growing murine cells were injected subcutaneously at the right side of the mouse in a volume of 200 μl of PBS. Animals were killed 3 weeks after tumor cell injection, and the tumors were processed for FACS sorting immediately. RNeasy microkit from Qiagen RNA concentration and purity from freshly sorted TAMs were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version 15031047 Rev.E) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR of 15 cycles was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. NextSeq 500 Experimental Factor: wild type genotype genotype Experimental Factor: low GLUT1 expression phenotype