B and IFNG Transcripts (Bottom Row) in the Top and Bottom 20% of EPHA2 Expression, in Human TCGA PDA Dataset

A B mRNA CD8A

C 8 R2=0.03705 6 p = 0.0098

2 mRNA IFNG -2 2468 -2 EPHA2 mRNA 2500 ** 800

2000 600 * 1500

400 mRNA 1000

200 IFNG 500

0 0 EPHA2 high low EPHA2 high low

D gMDSCs 80 ***

40 % CD45+ 20

0 T cell low high Figure S1. Expression of EPHA2 correlates with the abundance of CD8+ T cells in PDA (Related to Figure 1). (A) Top pathways from Metascape analysis of genes negatively correlated with CD8A in human TCGA PDA dataset. (B) Correlation of transcript abundance for CD8A and EPHA2 (left) and abundance of CD8A mRNA in the top and bottom 20% of EPHA2 expression (right) in human PDA samples (QCMG_Nature_2016 dataset, cbioportal). (C) Correlation of transcript abundance (top row) and abundance of CD3E, PRF1, GZMB and IFNG transcripts (bottom row) in the top and bottom 20% of EPHA2 expression, in human TCGA PDA dataset. (D) Flow cytometric analysis of immune cell populations in subcutaneously implanted T cell low and T cell high tumors derived from indicated clones (n=10/group). In (B-D), data presented as boxplots with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis by Students’ unpaired t-test (B, C, D) with significance indicated (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant in this and all subsequent figures, unless otherwise indicated). CD8+/CD11b+MHCII- C G

E CD8+/CD11b+MHCII- KO2 KO1 iueS.Tmrcell-intrinsic ( histogram Tumor Representative S2. Figure subcutaneous and and ouain nsbuaeu ( subcutaneous in populations 0gop.( 10/group).  yoer n36gop.( (n=3-6/group). cytometry 10/group) ifrnebtengop acltduigSuetsupie -et(B t-test unpaired Student’s using calculated groups between difference Ctrl IK WT A M n PDGFR and SMA Epha2- Epha2- 49561c 4956419c 6419c5 6419c5 6419c5 Arginase 1+ IDO1+ iNOS+ CD8+/myeloidcells CD8+/gMDSC CD8+/myeloid cells CD8+/gMDSC Macs Macs CD8+/gMDSC cells CD8+/myeloid Epha2 J Otmr rmidctdcoe n5gop.( (n=5/group). clones indicated from tumors KO Otmr rmidctdcoe.( clones. indicated from tumors KO rprin fK6+tmrclsaogcultured among cells tumor Ki67+ of Proportions ) 6419c5 Epha2 W and -WT  tiig nsubcutaneous in stainings W and -WT EPHA2 staining EPHA2 K-L A Epha2- n uniiain( quantification and ) C-D egt fipatdsbuaeu ( subcutaneous implanted of Weights ) 10 15 20 25 0 5 Epha2 =22/ru)adotooi ( orthotopic and n=12-25/group) , Otmr rmidctdcoe,2 asps mlnain Statistical implantation. post days 21 clones, indicated from tumors KO Epha2 *** K uosfo niae ln n1/ru) ( (n=10/group). clone indicated from tumors -KO 6694c2 JL D F 10 20 30 40 euae eliflrto Rltdt iue2). Figure to (Related infiltration cell T regulates H 0 Epha2 4956694c2 6419c5 B G-H Macs cDC CD103+ cDC CD103+ cDC Macs fEH2poenepeso nsubcutaneous in expression protein EPHA2 of ) Arginase 1+ IDO1+ iNOS+ lwctmti nlsso MSsadMc in Macs and gMDSCs of analysis cytometric Flow ) W and -WT 49561c 6419c5 6419c5 6419c5 5 % YFP+ B % F480+ Macs C-F Epha2 Epha2- lwctmti nlsso muecell immune of analysis cytometric Flow ) 0.0 0.5 1.0 1.5 2.0 2.5 E-F, D CD103+ cDC K 4956694c2 6419c5 =22/ru)adotooi ( orthotopic and n=12-25/group) , W and -WT -L). *** Otmr rmidctdcoe (n=4- clones indicated from tumors KO =-0gop uosfrom tumors n=5-10/group)

% F480+ Macs 100 20 40 60 80 0 Epha2- ** CD103+ (% CD45+)

Otmrcls yflow by cells, tumor KO CD103+ (% CD45+) Epha2 Epha2 1000 200 400 600 800 I uniiainof Quantification ) 10 20 30 40 0 CD206+ 0 -KO -WT 4956694c2 6419c5 *** *** Epha2 Epha2 L *** ,n=5- ( A-B -WT -WT ) Epha2 WT FDR Epha2 KO A FDR Translational initiation 0.010 DNA conformation change 0.006 Response to Type I Interferon 0.000 Cytoplasmic translation 0.009 Interferon gamma mediated signaling 0.000 0.000 Epithelial to mesenchymal transition 0.009 Response to Interferon gamma 0.000 Cell cycle phase transition 0.008 Cellular Response to Interferon gamma Defense response to virus 0.000 Multi organism metabolic process 0.008 Innate immune response 0.000 Sister chromatid cohesion 0.008 Negative regulation of Type I interferon 0.003 Transcription elongation from POI II 0.011 Cytokine mediated signaling 0.003 Establishment of protein to ER 0.011 Response to virus 0.005 RNA secondary structure unwinding 0.013 Response to interferon beta 0.005 Protein DNA complex organization 0.012 Negative regulation of viral process 0.009 DNA packaging 0.013 Regulation of translation initiation 0.015 0123 Normalized Enrichment Score 0.0 0.5 1.0 1.5 2.0 2.5 Normalized Enrichment Score B C D E 6419c5 80000 6419c5-Epha2-WT 6694c2-Epha2-WT 60000 40000

DAPI 20000 8000 6000 4000 STAT1 DESeq2 Scaled count Scaled DESeq2

2000 % YFP+ 0 1 0 1 4 44 l9 t3 x i c l1 fi35 Ifit1 as3 Ifi 1 1 p If c I Ifi44 M d27 k b Stat Cx O Isg15Isg20 D p Cx C Oas1a B2m Tap1 Tap2 2- 2- 6419c5-Epha2-KO 6694c2-Epha2-KO H H Ta 5000 6694c2 4000 80000 3000 6694c2 2000 60000 1000 40000 300 Epha2-WT 20000 200 8000 Epha2-KO 100 6000 4000 0 2000 1 l9 t1 3 1 4 5 0 a i44 c l10 i35 fi i44 fit 1 at If x c If I If I Mx 27 g1 g2 0 St C x Oas3 d Is Is as C C O 2 p k1 2m a - B Tap1 T 2 apbp F G H H2-D1T 6694c2 20000 **** WT PTGS2 padj value=0.0092 **** EPHA2-KO1 MACC1 padj value=0.0001 15000 **** EPHA2-KO2 CCK padj value=0.0000 **** 10000

5000

0 Ctrl IFN-g IFN-b

H I J K EPHA2 MFI

-2 -1 0 1 2 6694c2

6419c5

6499c4 EPHA2 MFI

2838c3

V 2 V 1 trl O E ctrl TGF E C -KO1 KO KO 3 3-KO 4-K d d3- d d a a ad3- ad4-KO1 a a Sm Sm Sm Smad3-KO2Sm Smad4-KO2 Sm Sm Figure S3. TGF-SMAD3, 4-PTGS2 axis as potential tumor cell-intrinsic regulator of TME (Related to Figure 4). (A) Gene set enrichment analysis of differentially expressed genes in Epha2-WT or Epha2-KO tumor cells using the GO biological processes gene sets (n=3-8/group). (B) Interferon responsive genes in tumor cells from implanted tumors (n=3-8/group). (C) Representative IF images of STAT1 staining (red) in tumors (n=3 /group). (D) Antigen presentation genes on implanted tumor cells (n=3-8/group). (E) Proportions of MHC I+ YFP+ tumor cells in implanted tumors (n=5/group). (F) MHC I protein expression on YFP+ tumor cells in vitro after 24 hours with either no treatment (ctrl) or IFN,orIFN treatments (n=3/group). (G) Differentially expressed genes identified in human T cell high (red) and T cell low (blue) PDA tumors (top and bottom 20% of CD8A expression). Genes with padj<0.05 and log2foldChange>2 shown. (H) Relative expression of Smad3 and Smad4 in control (EV) and KO tumor cell clones generated from 6419c5. Data from n=4 independent experiments. (I) EPHA2 protein expression in PDA tumor cell clones treated with either PBS orTGF for72hours.Datafrom n=3 independent experiments. Color key represents the normalized Z score.(J) EPHA2 protein expression on Smad3 and Smad4 KO cell lines in vitro (n=3/group). (K) EPHA2 protein expression in YFP+ cells from control and Smad3 and Smad4 KO tumors (n=5-9/group). In (E, H, K) data presented as boxplots with horizontal lines and error bars indicating mean and range, respectively. In (B, D, F and J), data presented as mean with error bars indicating SEM. Statistical difference between two groups calculated by Students’ unpaired t-test (E), between multiple groups determined by one-way ANOVA with Tukey’s HSD post-test (F, H, J, K). F A D rwh(ih) n(-)dt rsne sma iherrbr indicating bars error (F). R with in linear package mean and survival and (B-C) lme4 as the t-test using presented post-test unpaired HSD Students’ data Tukey’s using (B-C) calculated In differences (right). growth ( culture. in global 3 day on performed hs otat4 mgstkno a n a nclue(representative culture in 4 day and 1 day on taken ( images 4X contrast Phase ( (n=3/group). rndcd and transduced) extracellula and (B) expression mRNA eldpeincekb lwctmtyo a 5ps uo implantation. tumor post 35 day on cytometry flow by check depletion cell T iueS.Efc of Effect S4. Figure E Ptgs2-KO Ctrl i7 oto cr)and (ctrl) control Ki67+ ) Ptgs2 Day1 Day4 IgG anti-CD4/CD8 D ncot( knockout oto cr)and (ctrl) Control ) Ptgs2 Ptgs2 K (Cas9/ -KO Ptgs2 eeino D el nvtoadi io(eae oFgr 5). Figure to (Related vivo in and vitro in cells PDA on deletion Ptgs2 gO ie(=-0gop;tmrfe uvvl(et n tumor and (left) survival free tumor (n=5-10/group); mice -gKO) F oto cr)and (ctrl) Control ) Ptgs2 K el,sona ecn flv el;fo cytometry flow cells; live of percent as shown cells, -KO Ptges2 G2lvl C esrdi control in measured (C) levels PGE2 r K P el ltd500clswl n6wl plates. well 6 on cells/well 50000 plated cells KPC -KO E RAtasue)KCcl iecniindmedia conditioned line cell KPC transduced) gRNA Ptgs2 Ki67 1200

300 600 900 0 K P ellnsijce nW and WT in injected lines cell KPC -KO ctrl BC 102030400 120 160 40 80 0 Days post injection post Days ctrl Ptgs2 ie-fet oe with model mixed-effects ** Ptgs2 cr,Cs,n gRNA, no Cas9, (ctrl, ( -KO B-C -KO mg f3/group). of image E.Statistical SEM. Relative ) * Ptgs2 ( A ) utr rpeettv xeieto ,n3gop.( n=3/group). 2, of experiment (representative culture uniiain( quantification 6). of Figure Effect S5. Figure asidctn E AC n oposwt oiotllnsaderrbar error and lines Stud horizontal using calculated with prese differences boxplots Data Prism. Statistical square). and respect (E). (big respectively blue, (A-C) inset range, and the SEM in green indicating shown in area bars stained 20X nuclei cropped and the cells represents YFP+ fields/group). 25 CD3 C A DE YFP DAPI ( A-C E lwctmti nlsso oto cr)and (ctrl) control of analysis cytometric Flow ) fC3 el rd natctoostmr rmKC n KPCY and KPCY from tumors autochthonous in (red) cells CD3+ of ) B Ptgs2 CD73 MFI MHC I MFI eeino uo el n M nvtoadi io(eae to (Related vivo in and vitro in TME and cells tumor on deletion KPCY

% live cells 10 15 20 25 0 5 tl KO ctrl PD-L1+ **** D-E) Ptgs2 ersnaie2Xiae ( images 20X Representative Ptgs2 Control KPCY ns nardtts nGraphPad in t-test unpaired ents’ K uo el fe 8husin hours 48 after cells tumor -KO -KO Ptgs2 vl.Salsur n(D) in square Small ively. tda enwt error with mean as nted niaigma and mean indicating s Ptgs2 ie(n=20- mice D

and ) Pt g s2 ETcl ihtmr iho ihu h AC ramn.Tmrclsimpla cells Tumor treatment. GAFCP the starte without treatment or GAFCP diameter. (n=5-8/group). with tumor mm mice 3-5 tumors at C57BL/6 implantation high into cell subcutaneously T OE Rltdt iue7). Figure to cell-intrinsic (Related Tumor S6. Figure % change in tumor size % change in tumor size relative to baseline relative to baseline uo iecag eaiet h aeiei aetl(V and (EV) parental in baseline the to relative change size Tumor Ptgs2 rmtsrssac oimnteayi PDA in immunotherapy to resistance promotes

% change in tumor size relative to baseline aspost days 9 d Ptgs2 nted - Supplemental Table 1. Experimental Models: Tumor Cells and Mouse Strains

Tumor Cells Source

PENN 6419c5 Generated/B.Z.

Stanger

PENN 6694c2 Generated/B.Z.

Stanger

PENN 2838c3 Generated/B.Z.

Stanger

PENN 6499c4 Generated/B.Z.

Stanger

PENN 4662 Generated/R.H.

Vonderheide

Mouse Strains Source

KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre+/-; Generated

Rosa26YFP/YFP

KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre+/-; Generated/R.H.

Rosa26YFP/YFP;Ptgs2flox/flox Vonderheide

Tamoxifen-inducible Cre+/- Ptgs2flox/flox Generated/G.A.

FitzGerald Supplemental Table 2. Antibodies for Flow Cytometry

Target and clone Source Identifier

CD335 (NKp46) FITC (29A1.4) Biolegend 137606

CD279 (PD-1) FITC (29F.1A12) Biolegend 135214

FOXP3 PE (MF23) BD 560408

FOXP3 APC (FJK-16s) eBioscience 17-5773-82

CD8a PE-CF594 (53-6.7) BD 562283

CD8a APC-C7 (53-6.7) BD Pharmingen 557654

CD103 PE/Dazzle 594 (2E7) Biolegend 121430

CD103 PE-Cy7 (2E7) Biolegend 121426

CD223 (Lag-3) PE/Dazzle 594 (C9B7W) Biolegend 125224

CD3e PE/Cy5 (145-2C11) Biolegend 100310

CD11c PE/Cy5 (N418) Biolegend 117316

CD45 AF700 (30-F11) Biolegend 103128

CD8a PE/Cy7 (53-6.7) Biolegend 100722

I-A/I-E (MHCII) PE/Cy7 (M5/114.15.2) Biolegend 107630

CD44 V450 (IM7) BD 560451

Ly-6G V450 (1A8) BD 560603

H-2Kb/H-2Db (MHCI) AF647 (28-8-6) Biolegend 114612

Granzyme B AF647 (GB11) Biolegend 515406

F4/80 APC/Cy7 (BM8) Biolegend 123118

CD279 (PD-1) APC/Cy7 (29F.1A12) Biolegend 135224

Ki67 AF700 (B56) BD 561227

CD11b PerCP-Cy5.5 (M1/70) BD 550993

CD11b APC (M1/70) BD 553312

CD366 (Tim-3) PerCP-Cy5.5 (RMT3-23) Biolegend 119718

Nur77 PerCP-eFluor710 (12.14) eBioscience 46-5965-82

CD19 BV605 (6D5) Biolegend 115540 Supplemental Table 3. Antibodies for Flow Cytometry

Target and clone Source Identifier

CD11c BV605 (N418) Biolegend 117334

CD152 (CTLA-4) BV605 (UC10-4B9) Biolegend 106323

Ly-6C BV570 (HK1.4) Biolegend 128030

CD45 BV570 (30-F11) Biolegend 103136

CD4 BV650 (RM4-5) Biolegend 100546

CD8a BV785 (53-6.7) Biolegend 100750

CD90.1 BV650 (OX-7) Biolegend 202533

CD45.1 APC-Cy7 (A20) Biolegend 110715

Arginase1 PE (A1exF5) eBioscience 12-3697-82

IDO AF647 (2E2/IDO1) Biolegend 654004

eBioscience 53-5920-82 iNOS AF488 (CXNFT)

IL-10 FITC (JES5-16E3) Biolegend 505006

PD-L1 PE-Cy7 (10F.9G2) Biolegend 124314

PD-1 PE-Cy7 (RMPI-30) Biolegend 109110

IL-12 PECy7 (C15.6) Biolegend 505210

G-CSF eFluor660 (9B4CSF) ThermoFisher 50-7353-82

Scientific

GM-CSF PE (MP1-22E9) Biolegend 505406

NK1.1 APC Cy7 (PK136) Biolegend 108724

XCR1 FITC (ZET) Biolegend 148210

CD39 PE-Cy7 (Duha 59) Biolegend 143806

CD73 BV605 (Ty/11.8) Biolegend 127215

GR1 PE (RB6-8C5) Biolegend 108408 Supplemental Table 4. Antibodies for Flow Cytometry

Target and clone Source Identifier

SIRPa APC-Cy7 (P84) Biolegend 144014

CCR7 PE-Cy5 (4B12) Biolegend 120114

CD31 APC-Cy7 (ER-MP12) Abcore AC16-0061-

RORgT FITC (4G419) Abcam Ab104906

Caspase 3 activated FITC (A7R34) BD 550480

CD86 BV785 (GL-1) Biolegend 105043

GATA3 PE-Cy7 (L50-823) BD 560405

T-bet Pacific Blue (4B10) Biolegend 644808

B220 BV711 (RA3-6B2) BD 563892

CD64 PE-CF594 (145-2C11) BD 562286

Live/Dead Fixable Aqua Dead Cell stain ThermoFisher L34957

kit Scientific Supplemental Table 5. Gating Strategies

Gating Strategies

Tumor cells Live CD45- CD31-

Th1 Live CD45+ CD3+ CD4+ T-bet+

Th2 Live CD45+ CD3+ CD4+ GATA3+

Treg Live CD45+ CD3+ CD4+ FOXP3+

Th17 Live CD45+ CD3+ CD4+ RORgt+

Macrophages Live CD45+ F4/80+ CD11b+

cDC Live CD45+ F4/80- lineage- MHC II+ CD11c+

cDC1 Live CD45+ F4/80- lineage- MHC II+ CD11c+ xCR1+

cDC2 Live CD45+ F4/80- lineage- MHC II+ CD11c+ SIRPa+

MDSC Live CD45+ CD11b+ Gr1+

Neutrophils Live CD45+ F4/80- Gr1+

Myeloid cells Live CD45+ CD11b+ MHCII- or Live CD45+ CD11b+

gMDSC Live CD45+ CD11b+ MHCII- Ly6G+ Ly6C+ Supplemental Table 6. Antibodies for Immunofluorescent and Immunohistochemistry Stainings

Target and antibody dilution Source Identifier

CD3 Rabbit 1:50 Abcam ab5690

GFP Goat 1:500 Abcam ab6673

COX-2 rabbit monoclonal antibody 1:100 ThermoFisher RM-9121-R7

Scientific

SMA 1:100 Abcam ab5694

PDGFR 1:100 Abcam ab32570