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Supplemental Figures A Spleen Liver MLN B 750 Spleen MLN Liver/Tumor s cell 6 500 Control CFU-GM 10 BFU-E per CFU-GEMM 250 U-C CF 0 l l r er ro pa ro pa v ve or nt nt li li He He l tum Co Co ro pa pa nt He He Hepa Co ) C 3 D 2 5 * E 4 10 *** 10 ) * ** 4 Control CFU-GM (g 2 C( 8 e BFU-E 2 * 2 3 Hepa-2wk * Hepa-3wk 6 CFU-GEMM eight 2 CFU- hang 2 c Hepa-4wk ic * rw 1 4 1 ld 2 en Fo 2 0 pl 2 ls Tumo 0 2 -1 0 LSKLK Tota l wk wk wk ro wk wk wk 2 3 4 nt 2 3 4 pa pa pa Co pa pa pa He He He He He He ) 7 *** *** F 0.6 8 5 G 2 ) *** 6 *** 10 Control 2 Control (g 4 5 e 0.5 y( Hepa 2 Hepa it 4 ght i *** 2 ar 3 3LL 3LL 3 0.4 hang ul we 2 ll B16-F10 c B16-F10 0.2 2 2 ld 2 ce TRAMP TRAMP 2 1 Min/+ Fo Min/+ 0.1 1 Apc 0 Apc Spleen 2 -1 0.0 Spleen 0 2 LSKLK H I before sorting Fresh 12 hr Day 1 Day 2 Day 4 Day 6 Day 8 gated in CD45+lin- gated in CD45+lin- LK:31.0 LSK:3.7 GMP: 35.6 LK:1.7 LSK:98.3 LK:15.9 LSK:84.1 LK:34.4 LSK:65.6 LK:67.8 LSK:32.2 LK:90.1 LSK:9.9 LK:93.5 LSK:6.5 LK:97.0 LSK:3.0 BM c-Kit Sca-1 GMP: 3.2 GMP: 12.5 GMP: 77.8 GMP: 5.6 GMP: 43.4 GMP: 62.6 LK:14.7 LSK:1.6 GMP: 17.1 BM LSK CMP: 12.6 CMP: 28.8 CMP: 54.1 CMP: 18.1 CMP: 39.6 CMP: 30.0 CD34 Spleen CD16/CD32 c-Kit CD34 Sca-1 CD16/CD32 gated in CD45+lin- after sorting LK:1.2 LSK:98.8 LK:21.8 LSK:78.2 LK:33.6 LSK:66.4 LK:62.1 LSK:37.9 LK:89.3 LSK:10.7 LK:90.2 LSK:9.8 LK:96.3 LSK:3.7 LSK: 98.3 c-Kit BM GMP: 98.6 Sca-1 GMP: 3.1 GMP: 14.8 GMP: 67.5 GMP: 7.2 GMP: 27.7 GMP: 49.8 Splenic LSK CMP: 18.3 CMP: 25.7 CMP: 46.7 CMP: 28.0 CMP: 28.0 CMP: 39.4 CD34 LSK: 97.5 CD16/CD32 GMP: 94.9 Spleen c-Kit CD34 Sca-1 CD16/CD32 Supplemental Figure 1. Characteristics of cancer-induced splenic EMH. (A) Representative images of the histological detection of c-Kit+ progenitor cells in the spleens (left), non-cancerous liver lobes (middle) and mesenteric lymph nodes (MLN; right) of control or Hepa mice (N = 6–10 per group). Scale bar, 50 μm. (B) Numbers of CFU-C obtained with one million nucleated cells isolated from indicated tissues of control or Hepa mice (N = 6–10 per group; mean and s.e.m.). BFU-E: Burst-forming unit-erythroid; CFU-GM: Colony- forming unit-granulocyte/macrophage; CFU-GEMM: Colony-forming unit-granulocyte/erythrocyte/ macrophage/megakaryocyte. (C–E) Tumor weight (C), fold changes in splenic LSK and LK cell numbers (D), and CFU-C numbers in the spleens (E) of control or Hepa mice at different time points (N = 4 per group; mean and s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way or two-way ANOVA followed by the Dunn’s multiple comparison tests). (F and G) Spleen weight (F, left), total splenocyte numbers (F, right), and fold changes in splenic LSK and LK cell numbers (G) of control (N = 7), Hepa (N = 8), 3LL (N = 10), B16-F10 (N = 4), TRAMP (N = 2), and ApcMin/+ (N = 8) mice. Result are shown as mean and s.e.m. ***P < 0.001 (Kruskal-Wallis test followed by the Dunn’s multiple comparison test). (H) Gating strategy and the population purity after fluorescence-activated cell sorting (FACS). Numbers in plots indicate the proportions of gated cells. (I) Kinetics of BM and splenic LSK cells from Hepa mice to differentiate into CMPs and GMPs in vitro with SCF, TPO, and Flt3L in serum-free medium. Numbers in plots indicate the proportions of gated cells. Representative results from one of three independent experiments are shown. DEN + CCl B16-F10 ApcMin/+ Bleeding A 4 *** *** *** 20 20 15 15 *** *** *** + Control LSK + Control LSK + 15 15 Control LSK + Control LSK F F F Control GMP Control GMP F 10 Control GMP 10 Control GMP Min/+ CS CS CS DEN LSK B16.F10 LSK CS Apc LSK Bleeding LSK 10 10 Min/+ M- M- M- DEN GMP B16.F10 GMP M- Apc GMP Bledding GMP 5 5 %G 5 %G 5 %G %G 0 0 0 0 BM Spleen BM Spleen BM Spleen BM Spleen Csf2 ) B ) C D E Total splenocyte T cell B cell Myeloid cell 4 20 20 mL mL Control on 0.02 0.02 0.04 0.00 si pg/ ) pg/ 3 -6 15 15 es F( 0 e( pr as CS 2 x1 ex 10 10 le l( M- re ve 1 ti 5 leve 5 0.02 0.03 0.04 0.00 Hepa mG SF la ru -C 0 Re 0 Se 0 l l GM l ro pa ro pa ro pa GM-CSF nt He nt He nt He Co Co Co CD45 F Spleen Liver/Tumor Csf3 Csf3 25 20 on on ) ) si si 20 -6 -6 15 es es 0 0 pr pr 15 x1 x1 ex ex 10 l( l( 10 ve ve ti ti leve leve 5 la la 5 Re Re 0 0 l r ro pa ve or nt li He tum Co pa pa He He Supplemental Figure 2. GM-CSF-expressing LSK cells in the spleens of mice with different types of tumors. (A) The endogenous GM-CSF expression in LSK and GMP cells isolated from mice with hepatoma induced with DEN and CCl4 (Control: N = 3; DEN: N = 6), mice bearing B16-F10 melanoma (N = 4 per group), 16-week-old ApcMin/+ mice bearing intestinal neoplasia (N = 3 per group), or a model of blood loss- induced EMH (N = 4 per group). Data are showed as mean and s.e.m. ***P < 0.001 (two-way ANOVA followed by Dunnett's test). (B) GM-CSF levels in the sera of control (N = 5) and Hepa (N = 6) mice, determined by ELISA. Data are showed as mean and s.e.m. P > 0.05 (Student’s t-test). (C) The Csf2 mRNA expression, relative to Actb, in the spleens of control and Hepa mice (N = 7 per group; mean and s.e.m.). P > 0.05 (Student’s t-test). (D) GM-CSF release was measured in the supernatants of 48-hr cultures of splenic stromal cells from control or Hepa mice (N = 5–6 per group; mean and s.e.m.), by ELISA. P > 0.05 (Student’s t-test). (E) The GM-CSF expression in indicated splenic cell populations from control or Hepa mice evaluated by flow cytometry (N = 6 per group; mean and s.e.m.). (F) The relative Csf3 expression in the spleens of control and Hepa mice (left), or in the noncancerous liver and tumor tissues of Hepa mice. Values are normalized to Actb expression. Data are shown as mean and s.e.m. (N = 4–6 mice per group). P > 0.05 (Student’s t-test). A Gated in CD45+ B Sham SPx Sham SPx Sham CD11b+ 76.2 CD11b+ 71.6 SPx 40 + 30 5 SSC D4 CD11b 20 nC 7.09 7.17 %i 10 Monocytic Monocytic 54.7 55.1 0 PMN-like PMN-like c e yti ik c -l Ly6C ono MN Ly6G M P C Tumor CD11b+Ly6G-Ly6Chigh (monocytic) cells D E 1:2 1:4 1:8 l 21.3 17.3 17.5 Sham Arg1 l Nos2 1.5 SPx 30 * 0.10 0.008 leve leve s 0.08 on on 0.006 lysi Sham si 1.0 20 si ndex i ic 0.06 es es if 0.004 pr 23.6 19.0 19.2 0.04 * pr ex ex 0.5 pec 10 0.002 ve 0.02 ve Division %S SPx lati 0.0 0 0.00 lati 0.000 Re Re 1:21:4 1:8 x e x x m P m P m P CFSE ha S lon ha S ha S S a S S ff E F G H Serum-free medium Unstained BM Spleen Sham-1 Sham-2 SPx (pooled) Sham LSK GMP LSK GMP SPx Arg1 87.5% 87.0% 87.6% 78.6% p-STAT3 DCFDA Gr-1 t-STAT3 CD11b 95.5% Isotype 97.2% Sham p-p65 99.8% 99.9% 99.8% 99.6% SPx t-p65 Ly6G E-actin Ly6C PD-L1 BM Spleen 1:2 1:4 1:8 1:2 1:4 1:8 22.2 20.1 17.0 90.7 91.3 91.8 I RPMI 1640 + 10% FBS LSK LSK BM Spleen LSK GMP LSK GMP 31.1 20.5 18.8 91.9 90.7 91.5 25.8% 31.6% 22.5% 35.2% GMP GMP CFSE CFSE Ly6C Ly6G 69.5% 84.1% 64.9% 82.1% DEN + CCl4-induced hepatoma J +DGM-CSF SCF only +isotype (3Pg/ml) 92.3 92.4 9.95 CXCR2 Ly6G BM Spleen 1:2 1:4 1:8 1:2 1:4 1:8 15.7 13.1 14.2 85.8 88.0 75.4 CFSE Min/+ LSK LSK Apc mice K +DGM-CSF SCF only +isotype (3Pg/ml) 25.0 17.6 18.7 84.8 82.9 59.9 88.4 89.9 20.3 GMP GMP CFSE CFSE CFSE Supplemental Figure 3.
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