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Supplementary Materials and Methods Supplementary Materials and Methods: In silico analyses For prognostic analyses using gene expression signatures of particular cell types, the biostatistics software R (version 3.0.2) and several bioconductor packages were used (1-3). Genes differentially expressed by fibroblasts or pericytes (GEO accession number: GSE17014) (4) were assessed in the ovarian cancer patient data set (GEO accession number: GSE9899) (5). A false discovery rate of 5% was defined as the signature gene set and used to compute a score in each of the tumour samples using a previously described method (6). Since the score is an objective measure of the quantity of the particular cell type, samples with high and low levels of pericytes were identified based on the median pericyte score. The clinical significance of this pericyte signature was then computed by testing the statistical significance of survival difference in the form of a Kaplan-Meier plot using Cox proportional hazards model. The same procedure was used to identify the prognostic significance of the fibroblast- specific gene set (4). For enrichment analyses of related genes differentially overexpressed in the early versus late relapse patients, Gene Ontology terms enriched were tested among the overexpressed genes (70 genes, >2-fold, p<0.05) in the early relapse group. A Fisher's exact test was applied in combination with the classic algorithm from the topGO package using query terms from Ensembl (7, 8). Results were corrected for multiple testing according to Benjamini-Hochberg (p≤0.05, FDR≤5%) (9), revealing 70 genes as overexpressed (2-fold, p<0.05) in patients who relapsed early. Gene Set Enrichment Analysis was run in its java desktop application with data sets for GO biological processes and KEGG pathways, provided by MSigDB (v4.0.entrez.gmt). Default parameter settings were applied and results with FDR >0.25 were dismissed (10). Transduction with GFP and luciferase of OVCAR-5 cells and pericytes 1 A replication defective 3rd generation lentiviral vector promoted by co-transfection of pFUGW, HIV- 1 packaging vector and VSV-G envelope glycoprotein expressing GFP in pKs-CMV-GFP derived from pEGFP-Cl (Clontech Inc.) and firefly luciferase was used to transduce OVCAR-5 cells (to track metastatic spread) and pericytes (to track their survival) in-vivo. The packaging cell line HEK293T was used for virus production and 8ug/ml Polybrene (Sigma) was used as a transduction reagent. GFP+ cells were collected by sorting and expanded in culture for up to two passages. Immunohistochemistry and immunofluorescence Primary antibodies used for immunohistochemical analyses of tumour xenografts isolated at early and late time-points were rabbit polyclonal anti-GFP (Abcam; #ab290; 1:1000), mouse anti-human Ki67 (Dako; #M7240 [clone MIB-1]; 1:100), rabbit polyclonal anti-cleaved caspase-3 (Cell Signalling; #Asp175 [clone 5A1E]; 1:800), mouse anti-human/mouse αSMA (Dako; #M085129 [clone 1A4]; 1:100), mouse anti-human mitochondrial protein antibody (Millipore ; #MAB1273; 1:500) and rat anti- mouse CD31 (BD; #553370; 1:50). Secondary antibodies used for immunohistochemistry were corresponding species-specific biotinylated secondary antibodies from Vectastain™ Elite ABC Kits (Vector Laboratories, Inc.) for GFP, Ki67, cleaved caspase-3, anti-human mitochondrial protein and αSMA and biotinylated rabbit anti-rat secondary (Dako; #E0468; 1:300) for CD31. Other specific reagents used for immunohistochemistry were the tyramide signal amplification (TSA) kit (Invitrogen), the 3-Amino-9-ethylcarbazole (AEC) staining kit (Sigma Aldrich), trypsin (BD; Trypsin250; #15240) and CaCl2 (Merck; BDH; #10070). Primary antibodies used for immunohistochemistry analyses on patient biopsy samples on tissue microarray were mouse anti-human αSMA (Dako; #M085129 [clone 1A4]; 1:100), rabbit PDGFRβ (Abcam;#ab32570;1:100) and mouse anti-human CD34 (Dako; #15632 ; neat) with corresponding Vectastain™ Elite ABC Kits (Vector Laboratories Inc). Primary antibodies used for immunofluorescent co-localisation analyses were mouse anti-human Ki67 (Dako; #M7240 [clone MIB-1]; 1:100), mouse anti-human/mouse αSMA (Dako; #M085129 [clone 1A4]; 1:100), rabbit polyclonal anti-GFP (Abcam; #ab290; 1:1000), rat anti-mouse CD34 (Abcam; #ab8158; 1:50), rabbit polyclonal CD73 (Abcam; #ab71822; 1:50), rat anti-mouse Sca-1 (BD; 2 #557403;1:200), rabbit polyclonal Ki67 (Novocastra; #301119; 1:2000), mouse IgG1 CXCL12/SDF-1 (R&D systems; #MAB350;1:100), mouse IgG2a E-cadherin (BD; #610182;1:100) and mouse IgG1 Epcam (Cell Signalling; #2929; 1:500). Corresponding fluorescent secondary antibodies used were goat anti-rabbit/rat/mouse IgG1/mouse IgG2a AlexaFluor 488/555/568/647 (Invitrogen; 1:200). For immunohistochemical staining of xenograft tumour sections and patient biopsy tissue microarrays, harvested tissue sections were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Sections were then de-paraffinized and processed for antigen retrieval by incubation in a pressure cooker at 125°C for 3 minutes in 0.1M Tris/HCL pH 9.0 or 10mM citrate buffer pH 6.0. Sections were then blocked in 1% BSA for 60mins and incubated with the appropriate primary antibody overnight at 4°C followed by incubation with the corresponding secondary antibody for 1 hour at room temperature. The sections were then developed by DAB staining according to the manufacturer’s instructions and counterstained with haematoxylin. For CD31 staining, antigen retrieval was carried out by incubating for 20 mins in trypsin - CaCl2 (260mg CaCl2 and 50mg trypsin made up to 200mL with 0.05M pH 8.0 Tris buffer) at 37°C, stained with appropriate antibodies using the Tyramide Amplification kit according to manufacturer’s instructions and visualised with AEC substrate. For immunofluorescent staining of xenograft tumours, formalin-fixed sections were de-paraffinized and processed for antigen retrieval by incubation in a pressure cooker at 125°C for 3mins in 0.1M Tris/HCL pH 9.0 or 10mM citrate buffer pH 6.0. Alternatively, O.C.T (TissueTek) embedded tissue sections were fixed for 10 minutes in acetone at -20oC. Sections were then blocked in 1% BSA and incubated with the appropriate primary antibody overnight at 4°C followed by incubation with the corresponding fluorescent secondary antibody for 1 hour at room temperature along with DAPI. Sections were mounted in fluorescent anti-fade mounting medium (Invitrogen) and stored in the dark. 3 Invasion assays The invasion assay was performed in a 96-well format consisting of a Boyden chamber-like set up made up of two chambers separated by a filter membrane (8 μm polyethylene, Cultrex). p4 pericytes (2 x 104 cells in 100μl in serum-free media) were seeded into the lower well to act as a chemoattractant while GFP+OVCAR-5 cells (5 x 104 in 50μl serum-free media) were mixed well with growth factor reduced Matrigel (BD Biosciences) and pipetted into the upper chamber. The media in the lower chamber of the transwell was replaced with fresh epidermalization media (11) at the start of the assay and incubated at 37˚C in 5% CO 2 for 24 hours. Invasion was quantified by placing cell dissociation/Calcein AM solution in the bottom chamber, detaching the migrated cells from the filter. Calcein AM is internalized by the dissociated cells and intracellular esterases cleave the acetomethylester (AM) moiety. Fluorescent free calcein was then quantified to determine the number of cells that had invaded and plotted on a standard curve. Fluorescence was detected at excitation 540 nm and emission 610 nm using a BMG Labtech (Offenburg, Germany) POLARstar Optima fluorescent plate reader. Morphometric analyses Tumours sections derived from control and pericyte co-injected mice were analysed with the same threshold and results plotted as percent staining intensity per visual field (x10 or x20) and analysed by Metamorph (Molecular Devices, Inc.). For relapse correlation analyses, individual biopsy sections from patient tissue microarrays were plotted as percent staining intensity per visual field (x4) and also analysed by Metamorph. For calculating microvessel density (MVD) in the xenograft tumours, the number of microvessels per visual field (x20) was counted using a modification of the Cell Counter plug-in of ImageJ (NIH software). Microvessel pericyte coverage index (MPI) was calculated as the percentage of vessels per visual field (x60) that were covered by αSMA+ cells using the Cell Counter plug-in of ImageJ (NIH software). 4 For analysing vessel association of αSMA+ cells in the xenograft tumours, the number of CD34+ vessel associated and non-associated αSMA+ cells was counted per visual field (x60) using the Cell Counter plug-in of ImageJ (NIH software) and plotted as a percentage of the total number of αSMA+ cells per unit area. For analysing vessel association of αSMA+ cells in the patient biopsy tissue microarrays, the number of vessel associated and non-associated αSMA+ cells was counted per visual field (x40) using the Cell Counter plug-in of ImageJ (NIH software) and plotted as a percentage of the total number of αSMA+ cells per unit area. For all morphometric analyses on xenograft tumours, 2-3 random fields from 5-8 tumours per experimental group (from 3 independent experiments) were analysed. For all morphometric analyses on patient tissue microarray data, 3 random fields from 7 tumours per group (early, late and no relapse) were analysed for quantitation of CD34 and αSMA+ cells. For analysing patient biopsy tissue microarray samples, the morphometric ratio of the stained area to that of the total area was calculated using MetaMorph (Molecular Devices, Inc.) and was used to denote the measure of the protein expression. The clinical significance of protein expression was assessed by stratifying the samples into two groups based on the median expression level and visualized using Kaplan-Meier curves. Statistical significance was assessed using Cox proportional hazards model. For prognostic correlation with the probability of relapse, the relationship between a particular protein expression and pericyte score was visualized using scatter plots.
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