Supplemental Figure 1 Color Key A -2 0 2 B z-score 100% 75% 50% 25% 0% KC pan 1 WT pan 3 WT KC pan 3 WT pan 2 WT pan 1 WT KC pan 2 C Color Key D a: Brevibacterium f: Chlamydiales b: Brevibacteriaceae g: Chlamydiia -3 0 3 z-score c: Sphingobacteriaceae h: Chlamydiae d: Sphingobacteriales i: Mogibacterium e: Sphingobacteriia j: Oscillospira k: Methylobacteriaceae NML Control Microb.-entrained MΦ PDA PDA Patient Population Control Microb.-entrained MΦ + Myd88i E F Ctrl Abx 350 * 300 250 200 150 40X 100 Tumor weight (mg) 50 0 x Ctrl Ab Supplemental Figure 2 A KC WT B ** * Actinobacteria * ** Bacteroidetes Cyanobacteria Deferribacteres * Firmicutes Proteobacteria % Relative abundance TM7 Others Time(wks) 3 9 13 16 20 24 28 32 36 3 9 13 16 20 24 28 32 36 Alpha Diversity Measure C E 60 KC WT 40 20 B. pseudolongum B. animalis 60 5 KC WT 0 B. adolescentis 40% Rel. abundance 3 9 13 16 20 24 28 32 36 3 9 13 16 20 24 28 32 36 20 Age (weeks) B. pseudolongum B. animalis 5 0 B. adolescentis % Rel. abundance 3 9 13 16 20 24 28 32 36 3 9 13 16 20 24 28 32 36 F Age (weeks) Week 3 Week 9 Week 13 p=0.678 p=0.02 p=0.385 Time(wks) 3 9 24 20 13 16 D 28 32 36 Week 13 KC WT Firmicutes; Ruminococcus Firmicutes; Dehalobacterium Alpha Diversity Measure Firmicutes; Oscillospira Bacteroidates; Odoribacter Axis.2 [12.7%] Actinobacteria; Bifidobacterium Axis.2 [23.8%] Axis.2 [24.7%] Week 16 Bacteroidetes; Bacteroidales Axis.1 [80.8%] Axis.1 [65.4%] Axis.1 [49.6%] Actinobacteria; Bifidobacterium Week 16 Week 20 Week 24 Week 20 p=0.339 p=0.036 p=0.021 Firmicutes; Dehalobacterium Firmicutes; Mogibacteriaceae Bacteroidetes; Bacteroidales Actinobacteria; Bifidobacterium Week 24 Firmicutes Unclass Lachnospiraceae Actinobacteria; Bifidobacterium Axis.2 [17.9%] Axis.2 [44.3%] Axis.2 [23.5%] Week 28 Firmicutes; Candidatus Arthromitus Axis.1 [73.3%] Axis.1 [48.6%] Axis.1 [66.6%] Deferribacteres; Mucispirillum Proteobacteria; G Desulfovibrionaceae Week 28 Week 32 p=0.014 p=0.002p=0.002 Firmicutes; Turicibacter Firmicutes; Coprococcus Actinobacteria; Cortobacteriaceae Actinobacteria; Bifidobacterium Week 36 Alpha Diversity Measure Firmicutes; Turicibacter Bacteroidetes; Bacteroides Axis.2 [23.6%] Bacteroidetes; Rikenellaceae Axis.2 [27.8%] Actinobacteria; Bifidobacterium -6.0 -4.8 -3.6 -2.4 -1.2 0 1.2 2.4 3.6 4.8 6.0 LDA Score (log 10) Axis.1 [59.0%] Axis.1 [63.5%] Supplemental Figure 3 A B C D Chao1 Observed Shannon PD whole tree * * * * * * * * Alpha Diversity Measure 60 KC WT 40 20 B. pseudolongum E B. animalis F 5 Chao1 Observed 0 B. adolescentis % Rel. abundance 3 9 13 16 20 24 28 32 36 3 9 13 16 20 24 28 32 36 * * * * * * Age (weeks) Alpha Diversity Measure G H Shannon PD whole tree * * * * * * Alpha Diversity Measure Supplemental Figure 4 A *** * % Relative abundance *** B Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Gut 2% 27% 7% 18% 13% 22% 1% 1% 4% 56% 45% 54% 57% 72% 18% 56% 52% 46% PDA Acidobacteria Tenericutes Firmicutes Bacteroidetes TM7 Synergistetes Deferribacteres Verrucomicrobia OD1 Fusobacteria Cyanobacteria Unassigned Lentisphaerae Actinobacteria Thermi Proteobacteria Gemmatimonadetes C D PDA Gut Veillonella Enterobacteriaceae a: Brachybacterium Alcaligenaceae b: Dermabacteraceae Enterococcus c: Prevotella Streptococcus d: Streptophyta Lachnospiraceae-Uncl. e: Enterococcus Lachnospiraceae f: Lactobacillus Clostridiales g: Coprococcus Prevotella h: Lachnospiraceae Bacteroides i: Butyricicoccus Blautia j: Fusobacterium Dialister k: Fusobacteriaceae Ruminococcaceae l: Acorus Ruminococcus m: Escherichia Faecalibacterium n: Treponema Methylobacterium o: Spirochaetaceae Delftia p: Spirochaetales q: Mycoplasma Elizabethkingia Duodenum Caulobacteraceae r: Mycoplasmataceae Pseudomonas Pancreas s: Mycoplasmatales 1 Pt. 2 3 8 9 1 7 5 4 6 7 9 6 5 4 2 8 3 Supplemental Figure 5 A Firmicutes; Megamonas NML Firmicutes; Dorea PDA Bacteroidetes; Prevotella Firmicutes; Gallicola Firmicutes; Christensenella Firmicutes; Anaerotruncus Firmicutes; Mogibacterium Proteobacteria; Escherichia TM7; TM7 3 Proteobacteria; Unclass. Enterobac. Actinobacteria; Rothia Proteobacteria; Proteus Synergistetes; Pyramidobacter Firmicutes; Unclassified Veillonellacea Bacteroidetes; Paraprevotella Firmicutes; Oribacterium Proteobacteria; Erwinia Fusobacteria; Leptotrichia Synergistetes; Synergistaceae Proteobacteria; Oxalobacter Firmicutes; Unclass. Aerococcaceae TM7; TM7 3CW040 Unassigned Firmicutes; Christensenellaceae Firmicutes; Veillonellaceae Proteobacteria; Cardiobacterium Proteobacteria; Klebsiella Firmicutes; Veillonella Bacterioidetes; Parabacteroides Proteobacteria; Enterbacteriaceae -4 -2 0 2 4 4% LDA Score (log 10) 46% B ACE Chao1 Observed Shannon Simpson PD 1.00 p=0.06 * ** ** ** *** a: Brachybacterium b: Dermabacteraceae c: Prevotella d: Streptophyta e: Enterococcus f: Lactobacillus g: Coprococcus h: Lachnospiraceae Alpha Diversity Measure i: Butyricicoccus j: Fusobacterium NML PDA NML PDA NML PDA NML PDA NML PDA NML PDA Supplemental Figure 6 A Abx Repop. Analysis B Repop. Analysis Cre G12D 8 14 16 22 Germ free 6 14 p48 ;Kras Cre G12D Weeks of life p48 ;Kras Weeks of life C Control Abx 6% 17% 20 D **** 39% 81% 53% + 15 SSA SSC 10 %CD3 5 CD3 Viability SSC-W CD45 0 * Ctrl Abx 40 E Control Abx 3 40 14 16 30 180 **** **** 20 150 Ctrl 10 % MDSCs Abx CD11b 120 ns 49 8 64 6 0 90 Gr1 Ctrl Abx * * Control (pg/ml) 60 F 60 Control Abx + * Abx *** ns 25 40 30 40 20 0 CD8 % of CD3 a b a 1 5 S 8 67 18 42 E 0 + + P-1 IP-3 Cxcl Cxcl CD4 CD8 MIP-1 MI M CD4 RANT G H Control Abx Control Abx 80 ** + 60 * Ctrl 40 6% 26% SSC 40% 74% SSC Abx CD8 CD8 % CD38 20 0 IFN-γ CD38 CD4+ CD8+ Control Abx I *** J Control Abx 50 * Control Abx 15 ns + + 4 40 D 30 10 C f 9% 9% o 20 SSC 14% 47% 5 CD4 %FoxP3 SSC % 10 0 0 + + ICOS ICOS LFA-1 FoxP3 Ctrl Abx K L M N ns O ns P ns * ** ** 75 20 65 70 * * 20 *** * 80 * ** + + 60 + Ctl a 15 52 56 15 60 45 39 42 Abx 10 10 40 30 T cells 26 28 Abx + KPC Feces % % T cells % CD44 5 5 % CD44 20 % TNF- 15 13 % MDSCs 14 0 0 0 0 0 0 Ctrl Abx Ctrl Abx Ctrl Abx Ctrl Abx Ctrl Abx Ctrl Abx KPC Repop KPC repop KPC Repop Supplemental Figure 7 A B *** Control Abx Abx + **** Control αPD-1 Abx αPD-1 * Ccl24 Ccl24 50 ns *** * Control Masp2 Masp2 + 40 Hmgn1 aPD-1 Hmgn1 30 Csf1 Csf1 11% 12% 26% 33% T Cells) SSC + 20 Abx C8b C8b C4a C4a % CD44 10 Abx + aPD-1 Retnla Retnla CD44 (CD4 0 Mapk3 Mapk3 *** **** Defa-rs1 Defa-rs1 C Abx + p=0.06 Gnas Gnas Control αPD-1 Abx αPD-1 ns ** * Control Il22 Il22 25 Hc Hc + 20 aPD-1 15 Ifna1 Ifna1 7% 7% 15% 20% T Cells) SSC Stat1 Stat1 + 10 Abx H2-Eb1 H2-Eb1 % CD44 5 Abx + aPD-1 Itgb2 Itgb2 CD44 (CD8 0 Ly96 Ly96 l o *** x Irf7 Irf7 D Abx + tr n PD-1 Ab* PD-1 Control αPD-1 Abx αPD-1 a ns a * Mknk1 Control Mknk1 Co ns 60 6 * x +*** Control Map3k1 Map3k1 + + Abx * Tnf Tnf Ab aPD-1 40 4 Mapkapk5 Mapkapk5 1% 3% 1% 7% T Cells) SSC + Abx Nfkb1 20 Nfkb1 2 Ifit2 Ifit2 % CXCR3 Abx + aPD-1 % of CD3 0 Cxcr4 Cxcr4 CXCR3 (CD4 0 + + l Cfd Cfd o x CD4 CD8 tr * Pik3c2g Pik3c2g n PD-1 Ab * PD-1 Abx + a a Gnaq Gnaq E Co ns Control αPD-1 Abx αPD-1 x + ns ns * Control 80 **Iigp1 Iigp1 40 Ab Il5 + + Il5 30 aPD-1 60 * Gnb1 Ctrl Gnb1 13% 19% 17% 38% T Cells) Mapkapk2 SSC 20 40 Mapkapk2 + Abx Ccl11 Abx Ccl11 10 % LFA-1 % CD38 20 Abx + aPD-1 Ccl19 Ccl19 LFA-1 (CD8 0 0 Il15 Il15 + + l Ctrl CD4 CD8Rock2 o x Rock2 tr n PD-1αPD-1Ab PD-1 Irf1 Irf1 a a Co Abx Birc2 Birc2 Abx, xαPD-1 + Raf1 Raf1 -1.7 Ab Ccl2 Ccl2 -1.2 Tgfb3 Tgfb3 -0.8 Cd55 Cd55 -0.4 0 Tyrobp Tyrobp 0.4 Mafk Mafk 0.8 Map2k6 Map2k6 1.2 Cd40lg Cd40lg 0 1.7 Supplemental Figure 8 Color Key A B ea-KPC adv-KPC -2 0 2 z-score Bacteroides Akkermansia Parabacteroides Blautia Rikenella Oscillospira Desulfovibrio H&E; 5x Ruminococcus Coprobacillus Odoribacter Rikenellaceae AF12 Flexispira Mucispirillum Bifidobacterium Sutterella C Candidatus Arthromitus Prevotella WT ea-KPC Lactobacillus Allobaculum Bacteroidetes; Prevotella Deferribacteres; Mucispirillum Paraprevotella Unassigned Bacteroidetes; Paraprevotella Firmicutes; Erysipelotrichaceae WT 5 WT 6 WT 4 WT 1 WT 3 WT 2 Proteobacteria; Helicobacteraceae ea-KPC 7 ea-KPC 6 ea-KPC 3 ea-KPC 8 ea-KPC 2 ea-KPC 4 ea-KPC 5 ea-KPC 1 Bacteroidetes; Bacteroidales adv-KPC 4 adv-KPC 3 adv-KPC 2 Adv-KPC 1 Firmicutes; Allobaculum Firmicutes; Coprobacillus Alpha Bacteroidetes; Prevotellaceae Proteobacteria; Flexispira Diversity Firmicutes; Anaerotruncus F G Measure Actinobacteria; Bifidobacterium Firmicutes; Unclass. Lachnospiraceae p=0.009 PD Firmicutes; Lachnospiraceae * * * -4 -2 0 2 4 LDA Score (log 10) D WT adv-KPC Bacteroidetes; Prevotella Firmicutes; Allobaculum Unassigned Firmicutes; Coprobacillus Axis.2 [13.4%] Firmicutes; Erysipelotrichaceae Bacteroidetes; Paraprevotella Actinobacteria; Bifidobacterium adv-KPC Firmicutes; Anaerotruncus ea-KPC Firmicutes; Unclass. Lactobacillales WT Firmicutes; Coprococcus Firmicutes; Unclass. Lachnospiraceae Firmicutes; Lachnospiraceae Axis.1 [57%] -4 -2 0 2 4 LDA Score (log 10) E H ea-KPC adv-KPC Stage I/II Stage IV Proteobacteria; Helicobacteraceae Firmicutes; Phascolarctobacterium Bacteroidetes; Bacteroidales Bacteroidetes; Paraprevotellaceae Firmicutes; Mogibacteriaceae Proteobacteria; Alcaligenaceae Bacteroidetes; Elizabethkingia Synergistetes;Unclassified;Synergistaceae Tenericutes; Mycoplasmataceae Firmicutes; Veillonella Proteobacteria; Enterobacteriaceae Firmicutes; Streptococcus -4 -2 0 2 4 -5 -4 -3 -2 -1 0 1 2 3 4 LDA Score (log 10) LDA Score (log 10).