TIB6 B16F1 LLC EL4. n=2 n=7 n=9 n=6

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1 Shojaei et al., Supplemental Fig. 1 9 Mean Inhibition by anti-vegf (%) TIB6 n=2 n=7 n=9 n=6 Supplemental Figure 1 Differential growth inhibition by anti-vegf treatment. The percent of tumor growth inhibition was calculated on the basis of tumor volumes in anti- VEGF vs. control treated mice in each tumor type at day 14. We chose day 14 for analysis since in some experiments, some of the tumor bearing mice had to be euthanized. n indicates the number of independent experiments performed. In each experiment, five mice were treated with anti-vegf or control antibodies. Bars represent mean of tumor growth inhibition (%) ± SEM. Indicates that the difference between and or TIB6 tumors is significant (p<.5). Indicates a significant difference between (p<.5) and or TIB6 tumors.

2 Shojaei et al., Supplemental Fig. 2 Control TIB6 Supplemental Figure 2 Infiltration of GFP cells in refractory tumors. Immunofluorescent staining of CD31 (red) and GFP cells (green) in TIB6,, and tumor sections isolated from mice treated with control or anti-vegf antibodies as described in Fig.1a. The vast majority of GFP cells are devoid of CD31 expression, indicating that BMMNCs do not directly contribute to the tumor vasculature in our models.

3 Shojaei et al., Supplemental Fig. 3 Lethal Irradiation (I) Hematopoietic Repopulation Tail Vein Injection of GFPBM-MNCs Generation of Chimeric Mouse with GFP Hematopoietic Cells GFP Four Weeks (II) Priming Subcutaneous injection of, or cells. GFP GFP GFP GFP Matrigel 1 Days (III) Bone Marrow Chimeric Model Isolate GFP cells from the bone marrow of the primed mice, admix with and SC inject into C57BL/6 mice (IV) Tumor Chimeric Model Isolate GFP cells from the tumor of the primed mice, admix with and SC injection into C57BL/6 mice (V) Treatment with or control antibodies and analyzing tumor refractoriness Supplemental Figure 3 Strategies to investigate the role of BMMNCs in tumor refractoriness to anti-vegf treatment. To monitor kinetics of BMMNCs in tumor bearing animals, GFPBMMNCs were intravenously injected into lethally irradiated C57BL/6 mice (I). Next, the chimeric mice were primed by implantation of sensitive () and refractory ( and ) tumors in matrigel (II). GFP cells from both BM (III) or tumors (IV) of chimeric mice were isolated, admixed with cells and were then injected sc into C57BL/6 mice. Implanted animals were treated with anti-vegf or control antibodies and the role of BMMNCs in mediating tumor refractoriness to anti-vegf treatment was investigated (V).

4 Shojaei et al., Supplemental Fig. 4 a Control Control TIB6 Gr b CD11b Control Control TIB6 Gr CD11b Supplemental Figure 4 FACS profile representative of CD11bGr1 cells in the tumors and BM of mice bearing refractory or sensitive tumors. The myeloid compartments in mice bearing TIB6,, and tumors were analyzed using a FACS machine and monoclonal antibodies against CD11b and Gr1. Displays are the representative CD11bGr1 subset in the tumors (a) and the BMs (b).

5 Shojaei et al., Supplemental Fig. 5 a Frequency Among GFP Cells b CD19 CD9 CD11C VEGFR1 VEGFR2 TIB6 TIB6 Treated Control Treated Frequency Among GFP Cells CD19 CD9 CD11C VEGFR1 VEGFR2 TIB6 TIB6 Treated Control Treated Supplemental Figure 5 Multilineage analysis of GFP cells in the tumor and BM in mice bearing refractory or sensitive tumors. C57BL/6 mice were implanted with TIB6,, and tumors and were treated with anti-vegf or control antibodies as described. BMMNCs and tumor isolates were harvested from each mouse and were stained with antibodies against CD19 (B lymphoid), CD9 (T lymphoid), CD11c (dendritic) and also VEGF receptors (R1 and R2). Graphs represent the frequency of each subset in the tumors (a) and in the BM (b) compartments. indicates that the difference between and or TIB6 tumor bearing animals is significant (p<.5). Indicates a significant difference (p<.5) between and or TIB6 tumor bearing animals.

6 Shojaei et al., Supplemental Fig. 6 a 25 2 Weight (mg) TIB6 TIB6 b Treated Control Treated 35 CD11b Frequency Among GFP Cells Gr1 CD11bGr1 5 TIB6 Treated TIB6 Control Treated Supplemental Figure 6 The spleen is an alternative site of homing for CD11bGr1cells in mice bearing refractory tumors. C57BL/6-GFP chimeric mice were implanted with TIB6,, or tumors and were treated with anti-vegf or control antibodies for 17 days, as described. (a) The spleen of mice bearing refractory tumors was significantly (p<.5) larger than the spleen of mice bearing sensitive tumors (b) Splenocytes were harvested from each mouse using mechanical disruption and were treated with lysis buffer to remove red blood cells. Spleen cells were then stained with anti-cd11b and anti-gr1 antibodies and were then analyzed by FACS to investigate the frequency of CD11bGr1 cells. A significant increase (p<.5) in the frequency of CD11bGr1 cells in the spleen of mice bearing refractory tumor compared to the sensitive tumors was found. Indicates that the difference between and or TIB6 tumors is significant (p<.5). Indicates a significant difference (p<.5) between and or TIB6 tumors.

7 Shojaei et al., Supplemental Fig Whole BM 1 4 CD11bGr1 1 4 CD11b-Gr1- Gr CD11b Supplemental Figure 7 Purification of CD11bGr1 from the BM C57BL/6 mice of mice implanted with or tumors. BMMNCs were labeled with CD11b conjugated beads and passed through large-scale magnetic columns to isolate CD11b and CD11bfractions. An aliquot from each fraction as well as unsorted cells were stained with CD11b and Gr1 fluorochrome conjugated antibodies to ensure the purity of the population of interest in each subset.

8 Shojaei et al., Supplemental Fig. 8 Mean Tumor Volume (mm 3 ) a Cell Line Primed Mice BMMMNCs Treatment CD11bGr1 CD11b-Gr1- Matrigel CD11bGr1 Matrigel CD11b-Gr1- Control Days b Vascular Surface Area (%) Cell Line Primed Mice BMMNCs None CD11bGr1 CD11bGr1 CD11b-Gr1- CD11b-Gr1-.4 Supplemental Figure 8 (a) Myeloid cells isolated from mice primed with sensitive tumors fail to induce refractoriness to anti-vegf. The graph shows growth curves of tumors admixed with - or matrigel-primed, BM-CD11bGr1cells and treated with anti-vegf (n=5 per group). The experiments were performed at the same time as those shown in Fig. 3d. Therefore, the control is common to both figures. Tumor volumes were measured for up to 21 days after implantation, as described. (b) Induction of angiogenesis by CD11bGr1 cells is one the mechanisms leading to refractoriness to anti-vegf treatment. VSAs were analyzed in mice harboring admixture of and CD11bGr1or CD11b-Gr1- cells. Indicates significant difference (p<.5) when comparing admixture of and CD11bGr1 cells from or primed mice to admixture with CD11b-Gr1- cells isolated from the same primed animals.

9 a b Shojaei et al., Supplemental Fig. 9 TGF-B FIZZ1 Wfs1 TP 14A EMAP SULF-2 Extracellular matrix 2 CTGF TFPI JAM-2 XCP2 Ramp2 IL-1R2 ROR-alpha Ephrin B1 Thrombospondin-4 SPARC-like 1 Semaphorin A Angiogenic Receptors Adhesion Molecules Transmembrane Protein T3 T2 T1 B2 Sensitive B3 B1 L2 L3 L1 E1 E2 E3 Refractory Supplemental Figure 9 Gene expression profiles of refractory and sensitive tumors. (a) Unsupervised cluster analysis expression profile of refractory (: E1-3; : L1-3) and sensitive (TIB6: T1-3; : B1-3) tumors. Red and blue colors indicate up- and downregulated genes respectively. (b) Analysis of genes differentially expressed (p<.5, >2 folds) in both refractory tumors relative to sensitive tumors following treatment with anti- VEGF for 17 days. Genes potentially involved in the regulation of angiogenesis and/or hematopoietic properties of myeloid cells such as growth factors, cytokines and adhesion molecules are displayed. Cytokines/Receptors Myeloid Related Growth Factor/Secreted Molecules IGF2 HSP9A FGF18 ELM1 Ledgfa scavenger receptor type A Macrophage C-type lectin Pigr3 Macrophage SRT-1 Mast cell carboxypeptidase A MCP1 G protein-coupled receptor MIP2 ScyA7 IL-8R G-CSF IL-1R2 IL-1 inducible protein IL-1-beta ILIX Precursor Relative Expression Compared to and TIB6 Tumors Differential expression in Differential expression in

10 Shojaei et al., Supplemental Fig. 1 Level of Transcripts Relative to GAPDH Cell Line Source of Myeloid Cells.5 Angiogenin FGF-b FGF1 HGF HIF PDGFA PDGFB PDGFC PLGF VEGFA VEGFB VEGFC VEGFD None Supplemental Figure 1 Expression of angiogenic factors in sensitive tumors admixed with myeloid cells isolated from refractory- or sensitivetumor primed mice. Admixtures of tumor cells and myeloid cells were harvested from C57BL/6 mice which had been treated with anti- VEGF. Total RNA was extracted as described, and the expression levels of some of the known angiogenic transcripts including Angiogenin, bfgf, FGF1, HGF, HIF, PDGFA, PDGFB, PDGFC, PLGF, VEGFA, VEGFB, VEGFC and VEGFD were evaluated using Taqman PCR.

11 Supplemental Table 1. Sequences of primers in Taqman PCR Transcript Forward Primer Reverse Primer Taqman Primer VEGFA TGTACCTCCACCCTGCCAAGT TGGAAGATGTCCACCAGGGT CCAGCGAAGCTACTGCCGTCCAATT VEGFB GACGATGGCCTGGAATGT GGTACTTGGATCATGAGGATCTG CACTGGGCAACACCAAGTCCG VEGFC CCACTAAAAATATTGTTCCTGCAT TGATCACAGTGAGCTTTACCAAT CCACTAAAAATATTGTTCCTGCATTCATTTTTATAGCA VEGFD TTGACCTAGTGTCATGGTAAAGC TCAGTGAACTGGGGAATCAC ACATTTCCATGCAATGGCGGCT PDGFA CGTGTGACATTCCTGAACATACTA GTCCTCGGACACGGTTTT CAATGTGCGTGCGGTCTTTGTTCT PDGFB CAATGCCGGGCCTCG TCGCACAATCTCAATCTTTCTCA CTCGATGCCTGATTCGGACGGC PDGFC TTTGCTTGTAAAATGCTTAATATCG CCCAAAATAGCCACATTCTTTTA TTGATTCAAATAGTCACCACATAA CCTAGGCA Angiogenin ACCACCATTAGCGCCATGTT CATATTTACGTGCCATGAAGAA TAGGGTGGTGCACTCAGCGCCG HIF-1α GCCCCAGATTCAGGATCAGA TGGGACTATTAGGCTCAGGTGAAC CCTAGTCCTTCCGATGGAAGCACTAG ACAA HGF GGCCCACTCATTTGTGAAC CATCCACGACCAGGAAC ACACAAAATGAGAATGGTTCTTGGTGTCA PLGF GCAGTAGCCCGTGGACTT CGGTCCAGGTCTTCCACCAACTG ACACACACCCAGACTTGTATCGGTCA FGF-b CCTCTCAGAGACCTACGTTCAA GGAGGTCAAGGCCACAAT CGGTCCAGGTCTTCCACCAACTG FGF1 CTTGCCACAGGTCACAATG GCCTTACAAAGGGCTCATGT TGTGCAGATAACCTTTGCATCATTTGC GAPDH ATGTTCCAGTATGACTCCACTTCACG GAAGACACCAGTAGACTCCACGACA AAGCCCATCACCATCTTCCAGGAGCGAGA