SUPPLEMENTARY FIGURE LEGENDS

Figure S1: FASN inhibition with orlistat shows synergistic activity with gemcitabine in AsPC-1 cells. Clonogenic assay: Cells were seeded for individual gemcitabine and orlistat treatments or cells that were previously treated with different gemcitabine concentrations for 72 hours, were seeded and orlistat was added subsequently. Colonies were stained with crystal violet 0.4% after 30 days (A) and counted using Quantity One- 4.5.0 software. Mean values are plotted with Error bars representing standard deviation (B). Asterisks indicate statistical significance: * P ≤

0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Figure S2: Effect of orlistat on the lipid content of pancreatic cancer cells. (A) Nile red staining of intracellular lipid of PANC-1 cells after treatment with control or orlistat

100 µM for 96 hours. (B) Triglycerides contents of PANC-1 and AsPC-1 after orlistat treatment for 96 hours using adipogensis assay kit.

Figure S3: Platensimycin shows synergistic activity with gemcitabine in AsPC-1 cells. (A) Isobologram represents the combination index of the drug combination for gemcitabine and Platensimycin in sequential schedule at 90 % inhibition level. (B)

Combination index was calculated from MTT data using Compusyn software.

Figure S4: Lipid synthesis inhibitor C75 improves the effect of gemcitabine in pancreatic cancer cell lines. Isobologram represents the combination index of the drug combination gemcitabine and C75 in three different schedules; simultaneous, sequential and reverse sequential at 90 % inhibition level. Figure S5: Lipid synthesis inhibitor fatostatin improves the effect of gemcitabine in pancreatic cancer cell lines. Isobologram represents the combination index of the drug combination gemcitabine and fatostatin in three different schedules; simultaneous, sequential and reverse sequential at 90% inhibition level.

Figure S6: Albumin-conjugated palmitate does not rescue the anti-proliferative effects of gemcitabine. A panel of pancreatic cancer cell lines was treated with control or gemcitabine in the presence of albumin-conjugated palmitate or stearate (control).

Cell viability was determined by MTT assays 72 hour post incubation. All the results were compared to the respective viability in control or gemcitabine-treated samples by a two-way ANOVA and Bonferroni post hoc analysis. * P ≤ 0.05.

Figure S7: AdCM does not rescue the antiproliferative effect of gemcitabine on pancreatic cancer cells under the regular and stress conditions. Bar charts represent the relative cell viability under treatment with gemcitabine in the presence/absence of adipocyte-conditioned media (AdCM) in low glucose/glutamine/serum conditions.

Figure S8: Effect of AdCM on the lipid content of pancreatic cancer cells. (A)

Phase contrast and Nile red staining of intracellular lipid of differentiated 3T3-L1 cells before and after collecting the AdCM. (B) Relative triglyceride contents in PANC-1,

AsPC-1 and Capan-1 cells after incubation with control DMEN or AdCM, along with 5% charcoal stripped serum for 72 hours, determined by utilizing an adipogenesis assay kit.

* P ≤ 0.05, ** P ≤ 0.01.

SUPPLEMENTARY TABLES Table S1: Metabolic pathway enrichment in pancreatic ductal adenocarcinoma patients treated with gemcitabine (data pooled from TCGA).

Disease Pathway Pathway Enrichment Enrichment p stage database score for score for value complete clinical response progressive disease All Fatty Triacylglycerol and Ketone Body Metabolism Reactome 4.608 4.758 0.0212 Stage 2 Triacylglycerol and Ketone Body Metabolism Reactome 4.611 4.782 0.022 All Purine Metabolism Reactome 2.304 2.318 0.7613 Stage 2 Purine Metabolism Reactome 2.28 2.318 0.4132 All Metabolism of Nucleotides Reactome 3.156 3.202 0.6272 Stage 2 Metabolism of Nucleotides Reactome 3.144 3.247 0.2509 All Pyrimidine Metabolism Reactome 1.311 1.383 0.3558 Stage 2 Pyrimidine Metabolism Reactome 1.302 1.399 0.1942 All Reactome 2.791 2.818 0.6841 Stage 2 Glycolysis Reactome 2.817 2.827 0.8936 All Pentose Phosphate Pathway KEGG 2.105 2.14 0.5385 Stage 2 Pentose Phosphate Pathway KEGG 2.098 2.131 0.5842 All Amino Sugar and Nucleotide Sugar Metabolism KEGG 3.091 2.967 0.0703 Stage 2 Amino Sugar and Nucleotide Sugar Metabolism KEGG 3.084 2.979 0.1674 All Reactome 3.695 3.648 0.4823 Stage 2 Citric Acid Cycle Reactome 3.693 3.659 0.6452

Table S2: Patient Characteristics for Figure 1D.

Number Frequency

Age 40-49 2 5.1 50-59 10 25.6 60-69 10 25.6 70-79 14 35.9

Grade 1 1 2.6 2-2.5 11 28.2 3-3.5 10 25.6 4 17 43.6

Gender F 11 28.2 M 28 71.8

Table S3: Cell line gemcitabine responsiveness (IC50), FASN expression, and mutation status.

Gemcitabine responsiveness was determined by MTT assays. FASN expression was determined by real- FASN mRNA Kras status P53 status PI3K Reference Log [GEM dose, expression status Cell name M] (arbitrary units) p.R218_splice p.Y220C - (1) BXPC3 -8.161717 1.558 p.G12V p.A159V PIK3CG: CCLE CAPAN-1 -7.857298 1.466 p.T827R p.G12V - PIK3C2G: CCLE CAPAN2 -8.466228 0.638 p.P129del p.G12V p.C242R PIK3C2G: CCLE CFPAC-1 -8.621602 0.631 p.P129del p.G12D p.P151S PIK3C2G: CCLE HPAF-II -7.980053 1.160 3’UTR-del p.G12R p.R282W PIK3C2G: CCLE p.P129del; 3’UTR-del; PIK3C2A: HUP-T3 -8.451488 0.928 3’UTR-del MIA PaCa- p.G12C p.R248W - CCLE 2 -7.851706 0.737 p.G12D p.R273H; - (1) PANC1 -6.977984 1.035 p.R273C S2-013 -8.156269 0.692 p.G12D p.R273H - (2) S2007 -9.24382 0.445 p.G12D p.R273H - (2) p.G12D p.R273H - (2) SUIT2 -11.53165 0.558 T3M4 -8.387322 1.450 p.Q61H p.Y220C - CCLE PATu8902 p.G12V p.C176S PIK3C2G: CCLE -7.298259 1.154 mutations mutations p.P129del p.G12V - PIK3C2A: CCLE 3’UTR-del; p.L1150S; PIK3R1: Intron QGP-1 -6.103143 5.376 Insertion SW1990 -7.812479279 0.202 p.G12D - - CCLE p.G12D - PIK3C2G: CCLE p.N1365K Intron AsPC-1 -7.636576067 0.609 insertion DAN-G -9.20405 0.213 p.G12V - - CCLE time PCR analysis. Mutation status was determined from previous publications or Cancer Cell Line Encyclopedia (CCLE) database (https://portals.broadinstitute.org/ccle/home), as indicated.

1. Deer EL, Gonzalez-Hernandez J, Coursen JD, Shea JE, Ngatia J, Scaife CL, et al. Phenotype and genotype of pancreatic cancer cell lines. Pancreas. 2010;39:425-35. 2. Moore PS, Sipos B, Orlandini S, Sorio C, Real FX, Lemoine NR, et al. Genetic profile of 22 pancreatic carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4. Virchows Arch. 2001;439:798-802. Table S4: Orlistat responsiveness in pancreatic cancer cell lines. Cell Line Orlistat IC50 (μM)

PANC-1, 1019

AsPC-1, 990.1

HPAFII, 534.7

Capan-1, 690.9

S2-013, 22.79

S2-007, 417.3

PANC-89, 377.1

CFPAC-1, 213.7

MIA PaCa-2, 17.89

COLO357 3363

Table S5: Combination index of gemcitabine and orlistat in pancreatic cancer cell lines PANC-1, AsPC-1, Capan-1, and HPAF-II. AsPC-1 PANC-1 Capan-1 HPAF-II Drug E E ED ED ED ED ED ED ED ED ED ED combination D7 D9 90 95 75 90 95 75 95 75 90 95 5 0 Gemcitabine 1. 21. 0. and orlistat 2.5 4.7 3.3 90. 0.7 1.1 0.5 0.3 0.3 18 91 97 (simultaneously 94 15 39 576 37 96 05 60 40 7 7 6 ) 0. 0. Gemcitabine 0.2 0.3 0.1 0.1 0.1 0.0 0.1 0.0 0.0 0.0 19 10 then orlistat 53 25 59 50 65 61 44 58 38 34 2 1 0. 0. 2.6 124 Orlistat then 0.1 0.1 0.8 1.6 3.1 0.4 0.8 0.3 09 64 5E- 3.9 gemcitabine 20 49 23 87 59 80 01 47 6 7 6 3

Table S6: Combination index of gemcitabine and C75 in pancreatic cancer cell lines PANC-1, and AsPC-1.

AsPC-1 PANC-1 Drug ED7 ED9 ED9 ED9 combination ED75 ED95 5 0 5 0 Gemcitabine and C75 0.72 0.86 0.99 0.91 1.045 0.849 (simultaneously 0 1 2 9 ) Gemcitabine 0.36 0.41 0.47 0.60 0.819 0.501 then C75 0 7 0 8 C75 then 0.47 0.39 0.35 0.49 0.661 0.413 gemcitabine 4 3 3 8

Table S7: Combination index of gemcitabine and fatostatin in pancreatic cancer cell lines PANC-1, and AsPC-1. AsPC-1 PANC-1 Drug combination ED9 ED75 ED95 ED75 ED90 ED95 0 Gemcitabine and fatostatin 1.68 (simultaneously) 3.368 1 1.180 0.872 0.891 0.950 Gemcitabine then 0.97 2.38E- 7.77E- fatostatin 0.532 9 1.668 0.012 6 9 Fatostatin then 0.21 gemcitabine 0.358 7 0.174 0.255 0.061 0.024

Table S8: Combination index of gemcitabine and thapsigargin in pancreatic cancer cell lines PANC-1. PANC-

Drug combination 1 ED75 ED90 ED95 Gemcitabine and thapsigargin 0.666 0.398 0.285 (simultaneously) Gemcitabine then 0.246 0.077 0.035 thapsigargin Thapsigargin then 0.221 0.153 0.121 gemcitabine

SUPPLEMENTARY MATERIALS AND METHODS Immunohistochemistry, Tissue microarrays, and IHC scoring Immunohistochemistry was performed by utilizing Novalink Polymer Detection Kit, as per the manufacturer’s protocol. FASN (ImmTech, Inc, New Windsor, MD, USA),

CHOP, ATF4 and Cleaved Caspase 3 (Cell Signaling Technology) primary antibodies were utilized. We utilized the tissue microarrays (TMAs) made from paraffin blocks of formalin-fixed primary pancreatic tumor tissues from rapid autopsies. Large 2.0 mm cores were utilized to generate the tissue arrays. Each block also contained control specimens of uninvolved kidney and colon tissue as well as pancreas from non- cancerous donors. The tissue microarrays blocks were cut into 4 micron thick sections and mounted on charged slides. Immunohistochemical staining was scored by utilizing a composite scoring method. The intensity score was given by evaluating staining intensity of positive staining (0= none; 1=+weak; 2 = ++ intermediate, 3= +++ strong).

The proportion score representing the percentage of positively stained cell (0 =none; 1=

1-5%; 2= 6–25%; 3 =26–50% 4= 51–75% 5=76-100%). The overall FASN protein expression in each sample is expressed as histoscore, which is the multiplication product of the proportion (0–5) and intensity scores (0–3) and is between 0–15, with a maximum of 15. The staining score was evaluated by two independent observers and validated by a clinical pathologist.

Western blot analysis

Immunoblotting was performed as previously described (1). Proteins were detected by utilizing primary antibodies against FASN (FASgen, New Windsor, MD, USA), SREBP,

PERK, p-PERK, eIF-2, p-eIF-2, BiP, XBP1s, ATF4, CHOP, and BCL-2 (Cell signaling , Danvers, MA, USA). For loading control, membranes were probed with anti-beta

Tubulin antibody (clone E7; Developmental Studies Hybridoma Bank, The University of

Iowa).

Quantitative, real-time PCR analysis

For RNA isolation, we harvested the cells in TRIzol reagent ( ambion, Carlsbad, CA,

USA) as per the manufacturer’s instructions. RNA was quantified using Cytation 3

(BioTek, Winooski, VT, USA) and used for cDNA preparation by utilizing Verso-cDNA synthesis kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s guidelines. Quantitative real-time PCR analysis with gene-specific primers (Table) utilizing SYBR Green Master Mix (Applied Biosystems, Grand Island, NY, USA) was performed with the ΔΔCt method using an ABI 7500 thermocycler, as described previously (2). All quantifications were normalized to ACTB.

Adipocyte-conditioned medium preparation

3T3-L1 cells were seeded in 100 mm plates in the pre-adipocyte expansion medium

(90% DMEM and 10% Bovine Calf Serum). Forty-eight hours after the cells reached

100% confluence, cells were cultured with differentiation medium (90% DMEM, 10%

FBS, 1.0 µM Dexamethasone, 0.5 mM Methylisobutylxanthine (IBMX) 1.0 µg/mL Insulin

(Sigma-Aldrich Co., St. Louis, MO, USA)) for 48 hours. The differentiation medium was then replaced with adipocyte maintenance medium (90% DMEM, 10% FBS, 1.0 µg/mL

Insulin) that was changed every 48 to 72 hours. The cells were fully differentiated at 7 to

15 days after induction, as evidenced by lipid droplet formation. Finally, we added serum-free, glucose-free, and glutamine-free medium for 24 hours and collected the adipocyte-conditioned medium. The conditioned medium was centrifuged at 1,200 g for

10 min and used immediately or stored at −80°C.

Nile Red staining

We prepared stock solution of nile red (Sigma-Aldrich Co., St. Louis, MO, USA) in acetone (1mg/ml). Staining was carried out on live (unfixed) cells. The attached cultured cells were covered with PBS and the dye was added directly at a 1:1000 dilution. The cells were then incubated for 5 minutes and the excess dye was removed by rinsing briefly with PBS. Images were captured by immunofluorescence microscopy.

ROS assay

We prepared a stock solution of 2’, 7’ –dichlorofluorescin diacetate (DCFDA) (Sigma-

Aldrich Co., St. Louis, MO, USA) in DMSO (10 mM). Staining was carried on live cells.

Cells (1 X 106 cells) were seeded in 60 mm plates. After 24 hours, cells were treated with solvent control, gemcitabine 200 nM, orlistat 200 µM, or gemcitabine and orlistat combination for 24 hours. Cells were treated with H2O2 (2mM) for two hours as a positive control. After incubation, cells were washed twice with PBS then DMEM without serum. Cells were then incubated with DCFDA dye (100 μM) in a dark incubator for 30 min. Cells were washed with PBS, trypsinized, and then suspended in PBS. Samples were analyzed immediately using flow cytometry on BD Accuri C6 ((BD Biosciences,

San Jose CA). We used forward scatter and side scatter to identify the cell population, and the parameter FL1 for CDFDA. The software used for analysis is FACSDiva software v 8.0 (BD Biosciences, San Jose CA).

Cell surface staining

We seeded cells (1 X 106 cells) in 60 mm plates. After 24 hours, cells were treated with indicated treatments for 48 hours. After incubation, we harvested cells using Accutase and determined the total cell number. We washed the cells 4 times with 0.2% Tween 20 in PBS, and centrifuged at 400 g for 5 min. We blocked the non-specific binding with 5%

BSA for 15 minutes and washed 4 times with 0.2% Tween 20 in PBS and centrifuged at

400 g for 5 min. Then we incubated the cells with 2 μg/ml CD133 (BioLegend, San

Diego, CA, USA), CD24, EPCAM (BD Biosciences, San Jose, CA, USA) for 30 minutes.

For fixation, we added 100 µl 4% PFA to each tube for 15 minutes at 4°C. We washed again the cells 4 times with 0.2% Tween in PBS and centrifuged at 400 g for 5 minutes.

Finally, we suspended the cells in 500 μl ice-cold PBS containing 10% FCS, 1% sodium azide and analyzed with flow cytometry.

Sphere formation assay

We used the assay protocol as previously described (3). Briefly, to ensure a single cell suspension, we resuspended cells in 3 mL of cold PBS passed the cell suspension through 25 G needle three times. We confirmed a single cell suspension using hemocytometer. We seeded 5000 cells/well in a 6 well low attachment surface plate in stem cell medium (DMEM/F12 (Sigma-Aldrich Co., St. Louis, MO, USA) containing B27 supplement, FGF 10 ng/ml (Life Technologies, Inc., Grand Island, NY) , EGF 20 ng/ml, BSA 0.4% (Sigma-Aldrich Co., St. Louis, MO, USA) and penicillin/streptomycin

(Hyclone, Logan, Utah, USA). Cells were incubated in a humidified atmosphere of 5%

CO2 at 37°C for 5 days with indicated treatments. After 5 days, we counted the primary spheres, which were greater than 50 μm diameter. Then we collected the media containing spheres into centrifuge tube, centrifuged the tubes at 400 h for 5 min. we discarded the supernatant and resuspended the pellet in 300 μl of 0.5% trypsin/0.2

%EDTA. We disaggregated the spheres by passing the cells through a 25 G needle until a single cell suspension was produced. We neutralized the trypsin with double the volume of serum-containing media, and then centrifuged the cells at 400 g for 5 min.

We resuspended the pellet in 100 μL of ice cold PBS and placed the entire single cell suspension into a 6 well low attachment surface plate in stem cell medium and incubated in a humidified atmosphere of 5% CO2 at 37°C for 5 days. Cells were treated with indicated treatments and 5 days later, we counted the spheres that were greater than 50 μm diameter.

References: 1. Shukla SK, Gebregiworgis T, Purohit V, Chaika NV, Gunda V, Radhakrishnan P, et al. Metabolic reprogramming induced by ketone bodies diminishes pancreatic cancer cachexia. Cancer & metabolism. 2014;2:18. 2. Chaika NV, Gebregiworgis T, Lewallen ME, Purohit V, Radhakrishnan P, Liu X, et al. MUC1 mucin stabilizes and activates hypoxia-inducible factor 1 alpha to regulate metabolism in pancreatic cancer. Proc Natl Acad Sci U S A. 2012;109:13787-92. 3. Shaw FL, Harrison H, Spence K, Ablett MP, Simoes BM, Farnie G, et al. A detailed mammosphere assay protocol for the quantification of breast stem cell activity. Journal of mammary gland biology and neoplasia. 2012;17:111-7.