Supplemental Materials Fatty acid binding protein E-FABP restricts tumor growth by promoting IFNβ responses in tumor-associated macrophages Yuwen Zhang, Yanwen Sun, Enyu Rao, Fei Yan, Qiang Li, Ying Zhang, Kevin A. T. Silverstein, Shujun Liu, Edward Sauter, Margot P. Cleary, Bing Li Supplemental Figures and Tables Supplementary Figure 1, related to Figure 1. 1 Supplementary Figure 1 The development of tumors in WT and E-FABP-/- mice A, 0.5×106 (left panel) or 1×106 (right panel) E0771 cells were orthotopically injected into the mammary fat pad of WT and E-FABP-/- mice (n=9/group). Tumor size was measured at 3 day intervals. RMA lymphoma cells (0.2×106) (B) or MC38 colon tumor cells (0.2×106) (C) were respectively injected into the left flank of the WT and E-FABP-/- mice (n=9/group). Tumor size was measured at 3 day intervals. Lung metastasis nodules were measured after euthanization of mice on day 24 post tumor implantation. Data are shown as mean ± SD (*, P < 0.05 as determined by Student’s t test). Supplementary Figure 2, related to Figure 2. Supplementary Figure 2 Phenotype analysis of tumor-bearing WT and E-FABP-/- mice 2 Spleens (A) or inguinal lymph nodes (B) were collected from naïve mice or E0771-tumor bearing mice on day 24 after E0771 tumor implantation. Total cell numbers were counted with a hemocytometer after the lysis of red blood cells. C, E0771 tumors were collected from WT and E-FABP-/- mice on day 24 after tumor implantation. The percentage of CD11+F4/80+ TAMs was analyzed by flow cytometric staining. Data are shown as mean ± SD (*, p< 0.05; ** p<0.01 as determined by Student’s t test). Supplementary Figure 3, related to Figure 3. Supplementary Figure 3 Dynamic changes of E-FABP-expressing TAMs in the tumor stroma A, tumors from WT and E-FABP-/- mice were collected at the indicated time points after E0771 cell implantation and the percentage of CD11b+F4/80+Ly6C+MHCII+CD11c+ (Q2 subset) population in the TAMs was determined by flow cytometric staining. B, the Q2 subset of TAMs was separated from tumors and spleens by a flow sorter at the indicated time points post E0771 cell implantation and the relative expression levels of E-FABP and A-FABP were measured by real-time PCR. Analysis of fold changes of E-FABP and A-FABP in tumor Q2 cells compared to respective FABP in spleen Q2 cells. Data are shown as mean ± SD. 3 Supplementary Figure 4, related to Figure 4. Supplementary Figure 4 Analysis of GM-BMMs from WT and E-FABP-/- mice A, flow cytometric analysis of the expression of CD11b, F4/80, CD11c, and MHCII on GM- BMMs from WT and E-FABP-/- mice. E-FABP expression in GM-BMMs from WT and E- FABP-/- mice was analyzed by real-time PCR (B), western blotting (C) and confocal microscopy (D). E, analysis of enriched pathways of differentially expressed genes regulated by E-FABP in tumor-stimulated GM-BMMs by IPA. F, confirmation of gene expression results obtained from Affymetrix microarray by real-time PCR. Data are shown as mean ± SD (*p<0.05, ** p<0.01). 4 Supplementary Table 1, related to Figure 4 Table 1. The selected differentially expressed genes in macrophages regulated by E-FABP # ID Entrez Gene Name Log2 Fold* FDR Fabp5 fatty acid binding protein 5 (psoriasis-associated) -7.889 1.68E-11 Ms4a4c membrane-spanning 4-domains, subfamily A, member 4B -3.247 4.16E-02 Cxcl10 chemokine (C-X-C motif) ligand 10 -3.184 3.36E-02 Gm14446 interferon-induced protein with tetratricopeptide repeats 1 -2.974 3.17E-02 Ms4a4c membrane-spanning 4-domains, subfamily A, member 4B -2.826 3.00E-02 Ifit3 interferon-induced protein with tetratricopeptide repeats 3 -2.783 4.57E-02 Iigp1 interferon inducible GTPase 1 -2.742 2.01E-02 Iigp1 interferon inducible GTPase 1 -2.721 2.35E-02 Pydc4 pyrin domain containing 4 -2.71 3.14E-02 Pydc4 pyrin domain containing 4 -2.595 3.86E-02 Cmpk2 cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial -2.517 4.12E-02 Rsad2 radical S-adenosyl methionine domain containing 2 -2.516 3.58E-02 Ifit2 interferon-induced protein with tetratricopeptide repeats 2 -2.442 3.79E-02 Cmpk2 cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial -2.389 2.45E-02 Oasl1 2'-5'-oligoadenylate synthetase-like -2.36 2.45E-02 Rsad2 radical S-adenosyl methionine domain containing 2 -2.341 3.55E-02 Isg20 interferon stimulated exonuclease gene 20kDa -2.284 2.88E-02 Ifi44 interferon-induced protein 44 -2.273 3.20E-02 Ifit1 interferon-induced protein with tetratricopeptide repeats 1B -2.234 4.21E-02 Cxcl11 chemokine (C-X-C motif) ligand 11 -2.219 2.01E-02 Cd69 CD69 molecule -2.212 2.46E-02 Slfn4 schlafen family member 12-like -2.188 4.71E-02 Ms4a6b membrane-spanning 4-domains, subfamily A, member 6B -2.18 3.61E-02 Mx1 myxovirus (influenza virus) resistance 1 -2.129 2.45E-02 Tnfsf10 tumor necrosis factor (ligand) superfamily, member 10 -1.969 2.63E-02 Ctgf connective tissue growth factor -1.823 1.97E-02 Tnfsf10 tumor necrosis factor (ligand) superfamily, member 10 -1.795 2.09E-02 Tlr3 toll-like receptor 3 -1.794 3.65E-02 Mx2 myxovirus (influenza virus) resistance 1 -1.775 2.65E-02 Rsad2 radical S-adenosyl methionine domain containing 2 -1.719 4.37E-02 Ms4a6b membrane-spanning 4-domains, subfamily A, member 6B -1.702 4.05E-02 Ddx60 DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 -1.697 1.60E-02 Ms4a4b membrane-spanning 4-domains, subfamily A, member 4B -1.697 2.45E-02 Fam26f family with sequence similarity 26, member F -1.696 2.97E-02 Bambi-ps1 BMP and activin membrane-bound inhibitor, pseudogene (Xenopus laevis) -1.651 3.44E-02 Gbp6 guanylate binding protein 6 -1.647 2.98E-02 Tlr3 toll-like receptor 3 -1.643 2.95E-02 Irf7 interferon regulatory factor 7 -1.623 3.61E-02 Gm9706 predicted gene 9706 -1.6 3.97E-02 Nt5c3 5'-nucleotidase, cytosolic IIIA -1.538 2.47E-02 Ms4a6c membrane-spanning 4-domains, subfamily A, member 6C -1.517 4.39E-02 Igtp interferon gamma induced GTPase -1.502 3.73E-02 Ddx60 DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 -1.502 3.61E-02 Serpina3g serine (or cysteine) peptidase inhibitor, clade A, member 3G -1.502 1.84E-02 Gm11772 predicted gene 11772 -1.491 4.04E-02 Herc6 HECT and RLD domain containing E3 ubiquitin protein ligase family member 6 -1.471 3.92E-02 Ifi47 interferon gamma inducible protein 47 -1.462 3.60E-02 Gbp3 guanylate binding protein 4 -1.451 2.65E-02 Ddx4 DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 -1.45 2.18E-03 Ifnb1 interferon, beta 1, fibroblast -1.432 1.26E-02 Pyhin1 interferon activated gene 204 -1.421 3.79E-02 Herc6 HECT and RLD domain containing E3 ubiquitin protein ligase family member 6 -1.4 2.97E-02 Ly6a lymphocyte antigen 6 complex, locus A -1.387 4.58E-02 Herc6 HECT and RLD domain containing E3 ubiquitin protein ligase family member 6 -1.381 3.13E-02 Mxd1 MAX dimerization protein 1 -1.374 1.85E-02 Rilpl1 Rab interacting lysosomal protein-like 1 -1.369 1.60E-02 Hspa1b heat shock 70kDa protein 1A -1.358 1.66E-02 Gbp7 guanylate binding protein 7 -1.352 1.97E-02 Enpp4 ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative) -1.334 3.86E-02 Hspa1b heat shock 70kDa protein 1A -1.32 1.60E-02 Chrna5 cholinergic receptor, nicotinic, alpha 5 (neuronal) -1.316 1.60E-02 Gpsm2 G-protein signaling modulator 2 -1.303 3.30E-02 Pyhin1 interferon activated gene 204 -1.289 4.42E-02 Col1a2 collagen, type I, alpha 2 -1.287 2.62E-03 Stat2 signal transducer and activator of transcription 2, 113kDa -1.275 1.66E-02 Hspa1b heat shock 70kDa protein 1A -1.268 1.26E-02 (Table continues) 5 Table 1. (continued) # ID Entrez Gene Name Log2 Fold* FDR Irgm2 immunity-related GTPase family M member 2 -1.268 3.79E-02 Enpp4 ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative) -1.262 2.65E-02 Usp18 ubiquitin specific peptidase 18 -1.258 3.48E-02 Pou3f1 POU class 3 homeobox 1 -1.256 1.56E-02 Aldh1b1 aldehyde dehydrogenase 1 family, member B1 -1.247 3.61E-02 Herc6 HECT and RLD domain containing E3 ubiquitin protein ligase family member 6 -1.239 2.35E-02 Tnfsf10 tumor necrosis factor (ligand) superfamily, member 10 -1.234 4.93E-02 Stat1 signal transducer and activator of transcription 1, 91kDa -1.229 2.98E-02 Ifi203 interferon activated gene 204 -1.225 4.27E-02 Irgm1 immunity-related GTPase family, M -1.215 4.39E-02 Hspa1a heat shock 70kDa protein 1A -1.21 1.60E-02 Slfn5 schlafen family member 5 -1.206 3.79E-02 Daxx death-domain associated protein -1.201 3.02E-02 Ifih1 interferon induced with helicase C domain 1 -1.198 3.01E-02 Nlrc5 NLR family, CARD domain containing 5 -1.193 3.61E-02 Gbp2 guanylate binding protein 2 -1.158 2.91E-02 Stat2 signal transducer and activator of transcription 2, 113kDa -1.155 2.45E-02 Il15ra interleukin 15 receptor, alpha -1.137 2.09E-02 Rilpl1 Rab interacting lysosomal protein-like 1 -1.129 1.33E-02 Fcgr1 Fc fragment of IgG, high affinity Ia, receptor (CD64) -1.128 4.58E-02 Samhd1 SAM domain and HD domain 1 -1.127 1.60E-02 Gbp7 guanylate binding protein 7 -1.127 2.88E-02 Oas3 2'-5'-oligoadenylate synthetase 3, 100kDa -1.127 3.67E-02 Tpst1 tyrosylprotein sulfotransferase 1 -1.117 4.35E-02 Parp14 poly (ADP-ribose) polymerase family, member 14 -1.114 2.14E-02 Mxd1 MAX dimerization protein 1 -1.106 2.57E-02 Dcn decorin -1.1 2.04E-02 Xaf1 XIAP associated factor 1 -1.099 3.86E-02 Fabp3 fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) -1.095 2.72E-02 Stat1 signal transducer and activator of transcription 1, 91kDa -1.088 3.17E-02 Mthfr methylenetetrahydrofolate reductase (NAD(P)H) -1.083 3.61E-02 Il15 interleukin 15 -1.08 4.67E-02 Asb13 ankyrin repeat and SOCS box containing 13 -1.079 3.50E-02 Fabp4 fatty acid binding protein 4, adipocyte -1.078 4.38E-03 BC013712