Supplemental Information. Tissue-Resident Macrophages in Pancreatic. Ductal Adenocarcinoma Originate from Embryonic

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1 Immunity, Volume 7 Supplemental Information Tissue-Resident Macrophages in Pancreatic Ductal Adenocarcinoma Originate from Embryonic Hematopoiesis and Promote Tumor Progression Yu Zhu, John M. Herndon, Dorothy K. Sojka, Ki-Wook Kim, Brett L. Knolhoff, Chong Zuo, Darren R. Cullinan, Jingqin Luo, Audrey R. Bearden, Kory J. Lavine, Wayne M. Yokoyama, William G. Hawkins, Ryan C. Fields, Gwendalyn J. Randolph, and David G. DeNardo

2 Zhu et al. Figure S1 A Gated on live single cells CD CD11b Ly6G Ly6C F/8 F/8 B Orthotopic KPC Tumor Gated on live single cells CD CD11b Ly6G Ly6C F/8 F/8 C Orthotopic KPC Tumor Gated on CD + CD3 - CD19 - CD11b + Ly6G - Ly6C Low/- F/8 + + D Zbtb6 GFP/+ Reporter Mice Splenic DCs CD68 MerTK CD6 CD11 GFP B6 Control Zbtb6 gfp/+ Reporter Spleen CD8+ DCs E CX3CR1 Zbtb6 GFP/+ Reporter Mice CX3CR1-GFP CD6 SiglecF Isotype Control Antibody Stained PDAC Neutrophils 6 Zbtb6-GFP MFI Pancreatic DCs (CD+/Zbtb6 GFP +) Isotype Control Pancreatic DCs (CD+/Zbtb6 GFP +) Antibody Stained Pancreatic Macrophages (CD+/CD11b+/Ly6G-/Ly6C-/F/8++) Antibody Stained MerTK CD6 F/8 Orthotopic KPC Tumors MerTK CD6 F/8 CX3CR1 CD11

3 Zhu et al. Figure S A B Week of Parabiosis 6 Lymphocytes Monocytes Macrophages 6 Weeks of Parabiosis Lymphocytes Monocytes Macrophages Chimerism (% non-host derived) Chimerism (% non-host derived) 3 1 C Blood Pancreatic Tumor Blood Pancreatic Tumor -18 hours Clodronate i.v. Pancreatic Tumor Blood Pancreatic Tumor Blood Pancreatic Tumor hour Latex beads i.v. 1 hours hours 8 hours 7 hours Pancreatic Tumor Control w/o Beads CD11 CD11 CD CD11b Ly6C Beads-FITC Clodronate + Beads CD11 CD11 CD CD11b Ly6C Beads-FITC D Orthotopic KPC Tumor Gated on CD + CD3 - CD19 - CD11b + Ly6G - Ly6C Low/- F/8 + + Beads+ TAMs Total TAMs 1 Hours Hours 8 Hours 7 Hours F/8 F/8 F/8 F/8

4 Zhu et al. Figure S3 A Fmonocytes (% CD+ cells) 3 1 Ly6C Hi B6 CCR KO Nur77 KO Ly6C Low B6 CCR KO Nur77 KO B Steady-State Pancreas Macrophages (% of total cells) 8 6 n.s. CCR+/- CCR-/- C Macrophages (% of total cells) K KPC1 (Day 1) 1 1 Macrophages (% of total cells) K KPC1 (Day 1) 1 1 CCR-/- CCR+/- CCR-/- Macrophages (% of total cells) K KPC (Day 18) 1 1 B6 CCR+/- CCR-/- # of TAMs per pancreas (x1 6 ) K KPC1 (Day 1) 3 1 CCR +/- CCR -/- D K KPC1 (Day 1) K KPC1 (Day 1) K KPC1 (Day 18) K KPC (Day 1) CCR-/- CCR+/- CCR-/ CCR+/- CCR-/ B6 CCR-/ CCR+/- E F # of macrophages per pancreas # neutrophils per pancreas 1K KPC1 (Day 1) Vehicle αcsf1 + Clodronate Pancreas Neutrophils Day 1 8 IgG + PBS anti-csf1 + Clodronate # of macrophages per gram of pancreas # neutrophils per pancreas K KPC1 (Day 13) KPC-1 Orthotopic TAMs 3 1 IgG + Liposome anti-csf1 + Clodronate KPC-1 Neutrophils 1 Day 11 6 IgG + PBS n.s. anti-csf1 + Clodronate YFP-negative Macrophages per gram of tissue (x ) K KPC1 (Day 18) B6 KPC-1 Orthotopic in Flt3-Cre YFP Mice Normal Pancreas PDAC Nur77-/- Nur77-/- IgG + Liposome anti-csf1 + Clodronate G Photons / Second (x1 7 ) 3 1 K KC (Day 13).6... Day Day 7 Nur77+/- Nur77-/- Nur77+/- Nur77-/- Nur77+/- KPC- Orthotopic Tumor Burden (BLI) KPC-1 Orthotopic Tumor Burden Tumor Naive (Day ) Tumor-Bearing (Day 1) IgG + Liposome anti-csf1 + Clodronate

5 Zhu et al. Figure S E A C No PIMO Control 1-month-old Flt3-Cre YFP mice Gated on 9.8% 7.% YFP Macrophages YFP(-) TAMs No PIMO Control No PIMO Control Total TAMs YFP YFP(+) TAMs Orthotopic KPC-1 Tumor YFP PIMO PIMO PIMO B % YFP-negative of leukocytes Orthotopic KPC-1 TAMs YFP YFP D YFP-negative % of macrophage subsets F 8 6 PIMO MFI Hi 1 1 Low YFP-negative % of TAM subsets Orthotopic KPC Tumor 8 6 Hi Low Hi TAMs Low TAMs

6 Zhu et al. Figure S A Tamoxifen + progesterone one of the time points: E8., E9., E1., or E13. Csf1r-mer-iCre-mer; Rosa6-LSL-tdTomato 6 weeks Flow cytometry Kras-INK tumors 6 weeks ~1 days Flow cytometry B % tdtomato+ C % tdtomato+ of leukocytes E8. Pulse Blood Ly6C-Hi Monocytes Blood Ly6C-Low Monocytes PDAC Macrophages E8. E9. E1. E13. % tdtomato+ % tdtomato+ of leukocytes E9. Pulse Blood Ly6C-Hi Monocytes Blood Ly6C-Low Monocytes PDAC Macrophages Tamoxifen + progesterone gavage at E13. E8. E9. E1. E13. % tdtomato+ % tdtomato+ of leukocytes 7 E1. Pulse 6 Blood Ly6C-Hi Monocytes Blood Ly6C-Low Monocytes PDAC Macrophages PDAC Macrophages 6 E8. E9. E1. E13. % tdtomato+ of leukocytes E13. Pulse Blood Ly6C-Hi Monocytes Blood Ly6C-Low Monocytes PDAC Macrophages Control Cx3cr1-CreERT Rosa6-LSL-eYFP 6 weeks Macrophages Flow cytometry E13. Pulse.3% 9.% % YFP+ of leukocytes 1 1 YFP D Csf1r-mer-iCre-mer; Rosa tdtomato E8. Pulse 1 E Blood Ly6C-Hi Monocytes Blood Ly6C-Low Monocytes Cx3cr1-CreERT; Rosa YFP E13. Pulse % tdtomato+ of macrophage Subsets 8 6 % YFP+ of macrophage subsets 1 1 -Hi -Low -Hi -Low

7 Zhu et al. Figure S6 A B Blood Ly6CHi Monocytes -Hi -Low No BrdU control BrdU staining % Ki BrdU C IF- DAPI F/8 Merge Sirus Red (Collagen) 3x 3x Ki67 F/8 1 6 In St ro x m a Tu Ad m ja or C ce e nt ll 1 % Ki67+ of F8+ Cells 1 3 YF PP YF osi tiv Pe N eg at YF iv Pe Po YF si tiv Pe N eg at iv e Macrophage Proliferation on Collagen-I 1 % Ki67+ cells D 1 1 mg/ml 3 mg/ml Collagen-I

8 Zhu et al. Figure S7 A Gated on TAMs CD6 1 F/8 CD11b 1 MHCI 7 CD8 CD86 CD CD6 MFI 3 1 F/8 MFI 8 6 CD11b MFI 1 1 MHCI MFI 8 6 MFI 1 1 CD8 MFI 6 CD86 MFI 1 CD MFI PD1 MFI B D G % Fibrosis (TriChrome) 1 1 PD1 CXCR MFI 1 1 CXCR Gated on Macrophages CXCR post CD+ MACS enrichment K n.s. CXCR MFI 1 1 CX3CR1 MFI 3 1 YFP+ Macrophages CXCR YFP- Macrophages CX3CR CD1 MFI YFP+ Macrophages Identity Markers CAFs vs. TAMs CD CD11b MFI 8 6 YFP- Macrophages CD1 C Tie MFI 1 1 Gated on TAMs K 1K K K K 1K 1K K K K 1K 1K K K FSC-A SSC-H SSC-W CD CD3/CD19/B/Ly6G KPC- Orthotopic Analysis of Collagen Density Vehicle anti-cd11 (E13.) % Fibrosis (Sirus Red) 1 YFP- CCR+/- CCR-/- 7AAD/Ly6C F/8 F/8 PDGFRα Log 1 fold change (normalized to CAFs) Emr1 CAFs Itgam Csf1r Csfr CD11a YFP H I J CD11b Sort # Macrophage Identity Markers Ccr HSC-Derived TAMs (YFP+) Cx3cr1 E Embryonic-Derived TAMs (YFP-) Tie CD9d PDL1 MFI 1 1 Log 1 fold change (normalized to CAFs) Human PDAC GSE Sparc CD11a MFI PDL CD11a PDL MFI CD9d MFI TAMs vs CAFs- ECM and ECM Remodeling Tnc F Col1a 8 6 Sort # Has CAFs Cola Loxl1 PDL CD9d Embryonic-Derived TAMs (YFP-) Col1a Col3a1 Cola Cola1 Col6a1 Col1a1 Col17a1 Col18a1 Nid1 Tnc Eln Sparc Has Has3 Lox Loxl1 Adamts1 Mmp Mmp9 Yap1 Col6a Yap1 YFP+

9 Supplemental Figure Legends Figure S1, related to Figure 1. Macrophage Gating Strategy in and Orthotopic Tumors. (A) Representative flow cytometry plots showing gating strategy to identify macrophages in normal pancreas. (B) Representative flow cytometry plots showing gating strategy to identify macrophages in orthotopic tumors established using KPC-1 cells. (C) Measurement of listed surface markers analyzed by flow cytometry and pre-gated on macrophages in orthotopic KPC-1 tumors as shown in (B). To confirm CX3CR1 antibody staining, orthotopic KPC-1 tumors were established in CX3CR1 gfp/gfp mice; Representative flow cytometry plot of GFP signals in TAMs is shown. (representative of n=1). (D) Representative flow cytometry plots of Zbtb6-GFP fluorescence in TAMs of orthotopic KPC-1 tumors, brain microglia (CD Low /CD11b Hi /F/8 + ), and splenic dendritic cells (DCs) (CD + /CD11c + / + /B - /CD8 + or CD11b + ). Mean fluorescence intensity (MFI) for each cell population is calculated by deducting baseline MFI of isotype staining control from the MFI of stained samples. (n=-/group, mean ± SEM). (E) Measurement of listed surface markers in pancreatic dendritic cells (DCs) and macrophages by flow cytometry. Figure S, related to Figure. Substantial Portions of Macrophages in and PDAC Self-Maintain Independently of Blood Monocytes. (A-B) 3.-month-old homozygous CD.1 and CD. (KPC or wild-type C7BL/6) mice were surgically joined to create parabiotic pairs. Chimerism of indicated leukocytes were analyzed weeks (A) or 6 weeks (B) after the establishment of parabiosis. (n=6-16/group, mean ± SEM).

10 (C) KPC-1 orthotopic tumor-bearing mice were treated with clodronate i.v. to deplete circulating Ly6C Low monocytes, followed by retro-orbital injection of FITC-labeled latex beads. Representative flow cytometry plots showing FITC signals in blood Ly6C Hi monocytes hours after bead injection. (D) Representative flow cytometry plots of beads + TAMs in orthotopic KPC-1 tumors at indicated time points (n=3/time point). Figure S3, related to Figure 3. Deficiencies in Tissue Resident Macrophages but Not in Blood Monocytes Impact PDAC Tumor Burden. (A) Representative quantification of blood monocyte frequency in tumor-bearing B6, CCR -/- and Nur77 -/- mice. (n=3-/group). (B) Representative quantification of pancreatic macrophage frequency in tumor-free CCR +/- and CCR -/- mice. (n=3/group). (C) Orthotopic tumors were established in B6, CCR +/- or CCR -/- mice with, or, KPC-1 or KPC- cells. Frequencies or quantities of TAMs from independent experiments were analyzed at indicated time points after tumor implantation. Each bar graph represents an independent experiment. (D) Tumor burden analysis from (C). (E) Orthotopic tumors were established in B6, Nur77 +/-, or Nur77 -/- mice (numbers and cell types indicated). Tumor weights were measured at indicated time points. Each bar graph represents an independent experiment. (F) 8-week old C7BL/6 (left and middle) or Flt3-Cre YFP (right) mice were treated with αcsf1 combined with clodronate as described in Experimental Approach. Orthotopic tumors were subsequently established using KPC- or KPC-1 cells. Quantities of macrophages and neutrophils prior to or after tumor establishment were assessed by flow cytometry. (n=3-/data point) (G) Tumor burden from (F) were analyzed by bioluminescence imaging (BLI) or wet tumor weight. (n=-/data point/group) Data are shown as mean ± SEM and denotes p<. by t-test.

11 Figure S, related to Figure. Embryonically Derived Macrophages are Present in the Pancreas and PDAC of Adult Mice and are Enriched in the Low Subset. (A) Representative flow cytometry plots of YFP signals in pancreatic macrophages of 1-month-old tumor-free Flt3-Cre YFP mice. (B) Percentage of YFP-negativity in indicated leukocytes from 1-month-old tumor-free Flt3-Cre YFP mice. (n=3). (C) Representative flow cytometry plots showing YFP and signals in macrophages from normal pancreas of orthotopic KPC-1 tumors. (D) Percentage of YFP-negativity in Hi and Low macrophage subsets in normal pancreas and orthotopic KPC-1 tumors. (E) Representative flow cytometry plots of pimonidazole (PIMO) signals in indicated TAM subsets. (F) MFI of PIMO signals in indicated TAM subsets from (E). Data are shown as mean ± SEM and denotes p<. by t-test. Figure S, related to Figure. Yolk Sac and Fetal Liver Derived Pancreatic Macrophages are Present in the Pancreas and PDAC of Adult Mice and are Enriched in the Low Subset. (A) Csf1r-Mer-iCre-Mer; Rosa6-LSL-tdTomato mice were treated with tamoxifen at indicated time points. Labeling efficiency in indicated leukocyte populations was analyzed by flow cytometry. (n=3-7/group) (B) Kinetics of labeling efficiency in brain microglia, pancreas macrophages, and from (A). (C) Cx3cr1-CreERT; Rosa6-LSL-eYFP mice were treated with tamoxifen at E13.. Labeling efficiency in indicated leukocyte populations was analyzed by flow cytometry. Representative flow cytometry plots of YFP signals in pancreatic macrophages are shown.

12 (D) Labeling efficiency of CSF1R + cells in pancreatic macrophage subsets from (A-B). (E) Labeling efficiency of CX3CR1 + cells in pancreatic macrophage subsets from (C). Data are shown as mean ± SEM and denotes p<. by t-test. Figure S6, related to Figure 6. In Situ Proliferating Macrophages Localize in Proximity to Fibrotic Tumor Areas. (A) Representative flow cytometry plot showing 3-hour BrdU incorporation in blood Ly6C Hi monocytes. (B) Percentage of Ki67 positivity in indicated TAM subsets of KPC-1 tumor-bearing Flt3-Cre YFP mice. (C) Serial sections from autochthonous KPC tumors were stained for macrophages (F/8) and proliferation marker Ki67. Inlets demonstrate double positive cells. Fibrosis was assessed in the adjacent section by Sirius Red staining. Percentage of F/8 and Ki67 double-positive cells over total F/8 positive cells is depicted in areas proximal (< cell lengths) and distal from tumor cell nests (n=3/group). (D) Bone marrow-derived macrophages were cultured on collagen I at different densities. Percent of Ki67 positivity were quantified by immunofluorescence staining. Data are shown as mean ± SEM and denotes p<. by t-test. Figure S7, related to Figure 7. Embryonically Derived TAMs Have Distinct Surface Marker Expression and Unique Pro-fibrotic Transcriptional Profile. (A) MFI of indicated markers in TAMs from orthotopic KPC-1 tumors in Flt3-Cre YFP mice. Value is calculated by deducting baseline MFI of isotype staining control from the MFI of stained samples. (B) MFI of CXCR and in normal pancreatic macrophages. Representative histograms are shown. (C) MFI of CD11a and CD9d in TAMs from orthotopic KPC-1 tumors in Flt3-Cre YFP mice. Representative histograms are shown.

13 (D) Representative flow cytometry plots showing gating strategies used to sort YFP-positive and - negative TAMs from Flt3-Cre YFP mice. (E) Kaplan-Meier analysis of patient cohorts stratified by expression level of genes that are higher in Flt3-Cre YFP-negative TAMs based on analysis in Figure 7B. (F) Q-PCR analyses of indicated genes in Flt3-Cre YFP-positive (red) and Flt3-Cre YFP-negative (blue) TAMs in a second repeat of sorting. (n=/group for normal pancreas macrophages, n=6/group for TAMs). (G) Quantification of PDAC collagen density by image analysis of Trichrome or Sirus Red staining on orthotopic KPC- tumors implanted in mice treated with anti-cd11 at E13. or CCR -/- mice or control mice. (H) Expression of listed surface markers in cancer-associated fibroblasts (CAFs) (orange) and TAMs (pink). (I) Q-PCR analyses of indicated genes coding for macrophage surface identity markers in cancerassociated fibroblasts (CAFs) (orange), Flt3-Cre YFP-positive (red) and Flt3-Cre YFP-negative (blue) TAMs. (n=6/group for TAMs) (J) Q-PCR analyses of indicated genes coding for ECM production and modification molecules in cancer-associated fibroblasts (CAFs) (orange) and Flt3-Cre YFP-negative TAMs (blue). (n=6/group for TAMs) Data are shown as mean ± SEM and denotes p<. by t-test.

14 Table S1, Top Genes Higher in the Flt3-Cre YFP-Negative TAM subset on Microarray (Related to Figures 7 and S7) Entrez Gene ID Gene Symbol Fold Change (YFP- Adjusted p-value Negative vs. -Positive) 1699 Loxl Sfrp Fcer1a Cyp1b Col6a Fat Lum Serpina3n E Uncb E- 193 Tnc E- 183 Col6a Dcn Cald Sparc Dmbt Dlg Fermt Cpa Dkk Lox E Nid Sparcl Nfib Podn Lox Kirrel Rbp Yap Wfdc Bgn Pxdn Cola Mrc Pdgfrb Mal Foxg Adamts Col1a Tmsf Gm

15 QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analysis was performed using Unpaired Student s t-test, Mann-Whitney U test, Wilcoxon matched-pairs signed rank test, or ANOVA analysis as appropriate for the data set. Data in bar graphs are displayed as means ± SEM. Statistical significance is displayed as p<..