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SUPPLEMENTS Antihypoxic Effects of Myo-Inositol Trispyrophosphate on Vasculature and Therapy of Colorectal Liver Metastasis Perparim Limani1, Michael Linecker1, Philipp Kron1, Christoph Tschuor1, Andrea Schlegel1, Ekaterina Kachaylo1, Udo Ungethuem1, Jae Hwi Jang1, Stavroula Georgiopoulou2, Claude Nicolau3, Jean-Marie Lehn4, Rolf Graf1, Bostjan Humar1*, Pierre-Alain Clavien1* *shared senior authorship - Supplementary Methods - Supplementary Figure Legends Supplementary Methods Primary antibodies for immunohistochemistry antigen -catenin Caspase 3 cleaved Cd3 Cd31 Cd34 Cd45-B220 Cdh5 Cytochrome C F4/80 Hif1a Hif2a Ki67 Notch1 Piminidazole adduct pHistone 3 Vimentin company Novocastra Laboratories Ltd, Newcastle, UK Cell Signaling, Boston, MA Diagnostic Biosystem, Pleasanton, CA Abcam, Cambridge, MA LifeSpan BioSciences, Seattle, WA BD Biosystems, Franklin Lakes, NJ Abcam, Cambridge, MA Cell Signaling, Boston, MA BMA Biomedicals AG, Augst, Switzerland LifeSpan BioSciences, Seattle, WA Abcam, Cambridge, MA Cell Marque Lifescreen Ltd., Rocklin, CA Cell Signaling, Boston, MA Hypoxyprobe Plus Kit, Hypoxyprobe™, NPI Inc., Burlington, MA Abcam, Cambridge, MA Cell Signaling, Boston, MA order number NCL-B-CAT 9664 RMAB005 ab28364 LS-C180574 553084 ab33168 11940 T-2006 LS-B495 ab199 CMC27531021 36085 HP2-1000Kit ab92628 5741 Enzyme-linked immunosorbent assays (ELISA) ELISAs for Vegf (DY493), osteopontin (MOST00), Cxcl12 (MCX120), Scf (MCK00) and Plgf (MP200) were from R&D Systems (Minneapolis, USA), and Hif1a/Hif2a ELISAs (E90798Mu/ E93466Mu) from NewLife BioChemEX (Bethesda, MD). Triplicate measurements were performed. Western Blot Antibodies used were against lamin B1 (sc-377001, Santa Cruz Biotechnology, Dallas, TX) and βtubulin (2128S, Cell Signaling). Taqman gene expression assays Gene Arg1 Cd40 Cdh1 Cox4i1 Cpt1a Drp1 Emr1 Fis1 Hadha Hadhb Hif1a Hif2a Il6 Ldha Mfn2 Mgl1 Mrc1 Nos2a Ocln Opa1 Slc2a Snai1 Src Tnfa Twist1 Vegfa Vim Taqman assay no. Mm00475988_m1 Mm00441891_m1 Mm01247357_m1 Mm01250094_m1 Mm01231183_m1 Mm01342903_m1 Mm00802530_m1 Mm00481580_m1 Mm00805228_m1 Mm01217745_m1 Mm00468869_m1 Mm01236112_m1 Mm00446190_m1 Mm01612132_g1 Mm00500120_m1 Mm00546124_m1 Mm00443781_m1 Mm00440485_m1 Mm00500912_m1 Mm01349707_g1 Mm00441480_m1 Mm00441533_g1 Mm00436785_m1 Mm00443258_m1 Mm00442036_m1 Mm01281449_m1 Mm01333430_m1 All assays were from PE Applied Biosystems (Grand Island, NY). Ct values were normalized to 18SrRNA (TaqMan control reagents, PE Applied Biosystems) to represent relative fold induction of mRNA expression ± 95% confidence intervals. Tumor values were not normalized to unaffected liver, because tumor material originates from the intestine with a different basal expression pattern than liver. Supplementary Figure Legends Supplementary Figure 1. ITPP increases O2 dissociation from hemoglobin. A, O2 dissociation curve. ITPP shifts the dissociation curve to the right, reflecting the requirement of an increased pO2 to saturate hemoglobin with oxygen. Comparing the percentage in right shift following different administration routes (p.o, i.p., i.v.), intravenous injection was most effective. However, a drop in the right shift on day 2 by about 50% indicated weakening of the ITPP effects over time. Data are presented as means (n=3/group); error bars refer to ±95%CI. B, Immunoblot showing separation of cytosolic and nuclear fractions from tumor lysates. Subcellular fractionation was used to quantify by ELISA cytoplasmic and nuclear levels of Hifs. Supplementary Figure 2. Impact of ITPP on tumor burden, hypoxia and survival in the CT26/BALB model. A, Tumor response towards ITPP. Analysis included tumor development (representative MRI scans before and after treatment, ctrl=saline control), proliferation (Ki67, pH3, magnification 10x), apoptosis (caspase 3), necrosis inflammation (serum Hmgb1), and tumor volume/number (pathological and MRI assessment) immediately after treatment. B, Impact on tumoral Hif expression and hypoxia at day 17. Nuclear and cytoplasmic Hif levels (ELISA) and quantified pimonidazole staining for hypoxic areas immediately after treatment is shown. C, Kaplan-Meier survival analysis. Day 15 represents treatment end (n=5/group). Supplementary Figure 3. Impact of ITPP on metabolic and immune parameters in the CT26/BALB model at day 17. A, Impact on energy metabolism. Graphs show normalized gene expression in control tumors (light gray bars) and ITPP-treated tumors (dark grey bars). The upregulation of Cox4i1 to the expense of Slc2a and Ldha was less pronounced than in the MC-38/B6 model, but still is consistent with a switch from glycolysis towards oxidative phosphorylation following ITPP treatment. In agreement, the genes controlling mitochondrial dynamics are shifted from mitochondrial fission (Fis1, Dsp1) towards fusion (Mfn2, Opa and -oxidation genes (Cpt1a, Hadha, Hadhb) are upregulated in ITPP tumors compared to controls. Images (top right) show immunostaining for Cox4i1 protein in control (ctrl) and ITPPtreated tumors (magnification 40x). B, Impact on immunity. Graphs to the left show normalized expression of inflammatory (upper graph; Tnfa, Il6, and Emr1 encoding the macrophage marker F4/80) and macrophage polarization markers (lower graph; M1 markers: Cd40, Nos2a; M2 markers: Arg1, Mrc1, Mgl1). Images to the right (magnification 20x) show increased tumor infiltration of ITPP-treated tumors by macrophages (F4/80), B-cells (CD45B220), and NK cells (CD3). Supplementary Figure 4. Impact of ITPP on malignant tumor phenotype in the CT-26/BALB model at day 17. A, Immunohistochemistry for vimentin. Upper panels show control tumors with invasive spread into adjacent tissue (magnification 5x, 10x, 40x). Lower panels (magnification 100x) show fibroblastoid and spheroid morphology of untreated and treated cancer cells, respectively. B, Left: normalized gene expression of EMT inducers (Twist, Snai1, Src), a mesenchymal marker (Vim) and epithelial markers (Ocln, Cdh1) in control and ITPP tumors. Right: circulating levels of tumor-independent, systemic promoters of malignancy (Scf, osteopontin, Cxcl12) in control and ITPP-treated tumors. Supplementary Figure 5. Impact of oxygen levels and ITPP on cancer cells in vitro. One day after seeding MC-38 and CT-26 cells at hypoxia, cells were exposed or not to normoxia (A-C), or exposed to ITPP or saline (D-F) for 24h. A, Dependency of in vitro proliferation and mitochondrial activity on oxygen levels. Cells seeded at different densities (A=0.5x106, B=1x106, C=5x106) were exposed (5%O2) or not (1%O2) to normoxia. Cell numbers (growth) and mitochondrial activity (MTT assay) were assessed. The right graph shows the same set of experiments for CT-26 cells. B, Effect of normoxia on glucose consumption and lactate production. Glucose and lactate levels (glycolytic activity) were determined in supernatants from the experiments described under A (for the highest density only). C, Impact of normoxia on invasiveness. Single cell suspensions from hypoxic cultures seeded at 70% (cell density) and exposed or not to normoxia for 24h were assayed with Boyden chambers. D, Effect of ITPP on proliferation and mitochondrial activity. The same set of experiments described under A was performed for hypoxic MC-38 and CT-26 cells treated (+) or not (-) for 24h with ITPP (0.5mg/ml). E, Effect of ITPP on glucose consumption and lactate production. Supernatants from the experiments described under D were analyzed for glucose and lactate. All cell densities (A, B, C) were considered for the comparison between saline (-) and ITPP (+) to reveal potential ITPP effects. The patterned bars indicate glucose and lactate levels in fresh culture media. F, Effect of ITPP on in vitro invasiveness. Following a 24h exposure to saline (-) or ITPP (+), cells seeded at different densities were analyzed by Boyden chambers in analogy to C. Bars represent means and error bars 95% CI from triplicate measurements. Two independent replicates were performed. Supplementary Figure 6. Impact of ITPP on tumor vasculature in the MC-38/B6 model at day 17. Immunohistochemistry for Cd31, Cd34, Notch1 and VE-cadherin on liver samples immediately after treatment is shown. To better visualize vascular expression patterns, colors unrelated to the peroxidase-based staining were faded equally for all images using Adobe Photoshop. For Notch1/VE-cadherin, note the positive staining (Notch1 20x, upper corner) and (VE-cadherin, control, upper corner) in normal liver sinusoids, and the increases in both expression and the density of positive vessels within ITPP tumors. Supplementary Figure 7. Impact of ITPP on tumor vasculature in the CT-26/BALB model at day 17. Immunohistochemistry for Cd31, Cd34, Notch1 and VE-cadherin on liver samples immediately after treatment is shown. Fading of background color was applied for better visualization of staining patterns. For VE-cadherin/Notch1, note the increases in both expression and the density of positive vessels within ITPP tumors. Supplementary Figure 8. Long-term impact of ITPP on hypoxic response and tumor vasculature in the CT-26/BALB model. A, Long-term impact on the hypoxic response. Tumoral gene expression and serum quantifications are shown for control (-) and ITPP-treated mice (+) immediately (day 17) and one month (day 45) after treatment (see bottom for labels). Note the persistence of treatment effects for all parameters investigated. B, Long-term impact on tumor vasculature. Immunohistochemistry for Cd31, Cd34, Notch1 and VE-cadherin in control and ITPPtreated tumors at day 45. Fading of background color was applied for better visualization of staining patterns. Please compare with Supplementary Fig. 7B to note the similar expression patterns immediately and one month after ITPP treatment.