Chapter 3 Bone Marrow–Derived Vascular Progenitors and Proangiogenic Monocytes in Tumors

C H A P T E R

T H R E E

Bone Marrow–Derived Vascular Progenitors and Proangiogenic Monocytes in Tumors Kan Lu,* Chrystelle Lamagna,* and Gabriele Bergers*,† Contents 1. Introduction 2. Methods for the Visualization of BMDCs in Tumors 2.1. Transplantation of GFP-expressing bone marrow to visualize BMDC in tumors 2.2. Visualization of GFPþ BMDC subpopulations in tumors 2.3. Quantification of GFPþ BMDC subpopulations in tumors 3. Analysis of Pericyte Progenitors in Tumors 3.1. Detection of pericyte progenitors and pericytes in tumors 3.2. Isolation of PDGFRbþ pericytes from Rip1Tag2 tumor cell suspension 3.3. In vitro differentiation of pericyte progenitors References

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Abstract In tumors, new blood vessels develop not only from pre-existing vessels (angiogenesis), but can also be comprised of circulating vascular progenitor cells originating from the bone marrow (vasculogenesis). Besides endothelial progenitor cells (EPC) and pericyte progenitor cells (PPCs) that are incorporated into the growing vasculature, other subpopulations of bone marrow–derived cells (BMDC) contribute indirectly to tumor neovascularization by providing growth factors, cytokines, and other key proangiogenic molecules. Here, we describe specific methods that allow for the identification and functional characterization of these distinct BMDC populations in tumors as exemplified in mouse models of pancreatic neuroendocrine tumors and glioblastomas.

* {

Department of Neurological Surgery, Brain Tumor Research Center, University of California-San Francisco, San Francisco, California Comprehensive Cancer Center, University of California-San Francisco, San Francisco, California

Methods in Enzymology, Volume 445 ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)03003-6

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2008 Elsevier Inc. All rights reserved.

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1. Introduction A growing body of evidence supports the notion that new blood vessels do not exclusively originate from the existing vasculature by activation of vascular cells (endothelial cells and pericytes) within injured tissues or tumors, but are also formed with the help of vascular progenitors and proangiogenic myelocytic cells that are recruited from the bone marrow to sites of active vascular remodeling in the adult. Vascular progenitor cells consist of endothelial cell progenitor cells (EPCs) and pericyte progenitor cells (PPCs) that structurally contribute to the formation of new blood vessels. While EPCs incorporate into the vessel wall and mature into endothelial cells, PPCs envelop blood vessels and differentiate into pericytes supporting stabilization and maturation of the newly formed vasculature (Aghi and Chiocca, 2005; Allt and Lawrenson, 2001; Asahara et al., 1997; De Palma et al., 2005; Dome et al., 2008; Lyden et al., 2001; Rafii et al., 2003; Rajantie et al., 2004; Song et al., 2005). The ontogeny of vascular progenitors in the adult has become more complex with the revelation that vascular progenitors can originate from mesenchymal as well as from hematopoietic cells. The predominant portion of EPCs that are found in growing tumor vessels are derived from the bone marrow as CD45– VEGFR2þ CD133þ c-kitþ cells (Asahara et al., 1997; Rafii et al., 2002), but hematopoietic sources such as myeloid progenitor cells have also been described as having the propensity to differentiate into endothelial-like cells (Bailey et al., 2006; Rohde et al., 2006; Yang et al., 2004). Interestingly, in tumors bone marrow–derived (BMD) pericyte progenitors thus far appear to be of hematopoietic origin. BMD pericytes were identified as CD11bþ NG2þ cells in a subcutaneous Bl6-F1 melanoma model (Rajantie et al., 2004), and were found to originate from CD45þ Sca-1þ PDGFRbþ hematopoietic cells in a transgenic mouse model of pancreatic islet tumorigenesis (Song et al., 2005). In the latter tumor model, bone-marrow transplant experiments combined with FACS and immunohistochemical analyses revealed that BMD-Sca-1þ PDGFRbþ cells were able to develop into mature pericytes expressing the markers NG2, a-SMA, and desmin in vivo. Interestingly, only 15 to 20% of the pericytes in these tumors expressed the mature pericyte markers, whereas mature pericytes were devoid of PDGFRb. Complementary co-culture experiments of BMD or tumor-derived Sca-1þ cells with endothelial cells in a three-dimensional (3D) Matrigel matrix confirmed the propensity of these cells to differentiate into pericytes in vitro (Song et al., 2005). In addition, mesenchymal Sca-1þ Tie2þ CD13þ pericyte progenitors were identified in some tumors; however, these cells did not originate from the bone marrow but rather appeared to be recruited from the tumor stroma or neighboring tissue (De Palma et al., 2005). These cells are more reflective of

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pericytes that develop during vasculogenesis in the embryo, as those, albeit not exclusively like endothelial cells, originate from mesenchymal stem cells (Betsholtz et al., 2005; Carmeliet, 2003; Lamagna and Bergers, 2006). The extremely diverse incorporation rates of EPCs in blood vessels of different tumors, and even of the same tumor type at distinct stages of progression, have raised intense debate about the functional significance of EPCs in tumor neovascularization. Certainly in most instances, the incorporation rate of EPCs, and also of PPCs, is rather low but one cannot exclude the possibility that these cells can act as transient but significant catalysts of tumor neovascularization and progression. Congruent with this hypothesis, vascular-disrupting agents that ablate tumor blood vessels within a few hours and thereby cause severe hypoxia and necrosis, have been found to trigger transient homing of EPCs to the tumor margin, an effect sufficient to facilitate neovascularization and tumor regrowth (Shaked et al., 2006). Notably, these tumors did not contain substantial numbers of BMD cells (BMDCs) prior to treatment, but recruited such cells when confronted with a drug that scrutinized the tumor vasculature and caused hypoxia. The largest and most heterogeneous group of BMDCs in tumors consists of CD45þ myelocytic cells that contribute indirectly to neovascularization by expressing a variety of proangiogenic cytokines, growth factors, and proteases (Grunewald et al., 2006). Such cells include tumor-associated macrophages (TAMs) (Condeelis and Pollard, 2006; Pollard, 2004), immature monocytic cells including Tie2þ monocytes (TEMs) (De Palma et al., 2005), CXCR4þ VEGFR1þ hemangiocytes (Hattori et al., 2002; Jin et al., 2006), and Gr1þ CD11bþ myeloid cells (Bunt et al., 2006; Yang et al., 2004). Inhibition of any of these monocytic subpopulations in tumor models reduced or restrained tumor neovascularization (De Palma et al., 2005; Du et al., 2008; Grunewald et al., 2006; Pollard, 2004; Yang et al., 2004). Recent data revealed that one of the proangiogenic factors commonly expressed among these monocytic subpopulations is the matrix metalloproteinase MMP-9 (Ahn and Brown, 2008; Bergers et al., 2000; Coussens et al., 2000; Du et al., 2008; Seandel et al., 2008; Yang et al., 2004). MMP-9 can promote neovascularization by different means. It degrades extracellular matrix components to allow endothelial cell invasion and cleaves c-kit ligand to facilitate mobilization of vascular progenitor cells from the bone marrow into the bloodstream (Egeblad and Werb, 2002; Heissig et al., 2002; Page-McCaw et al., 2007). In addition, MMP-9 has also been shown to be essential in initiating neovascularization in mouse models of pancreatic islet tumorigenesis and glioblastomas whereby MMP-9 expressed from tumor-recruited BMDCs can liberate sequestered VEGF from the extracellular matrix, thus increasing VEGFR-2 activation and angiogenesis (Bergers et al., 2000; Du et al., 2008). Importantly, CD45þ cells that expressed MMP-9 were shown to be sufficient to initiate the angiogenic switch in pancreatic and brain tumors.

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How do BMDCs specifically home to sites of vascular remodeling in tumors? Thus far only a few factors are known to enable the mobilization of BMDC from bone marrow into the bloodstream, as well as their subsequent recruitment and retention into the tumor. The most prominent factors identified to date include tumor-produced VEGF and PlGF, which act as mobilization factors of EPCs, and PDGF-B, which mobilizes PPCs (Gerhardt and Betsholtz, 2003; Gerhardt et al., 2003; Li et al., 2006; Luttun et al., 2002; Rabbany et al., 2003); and stromal-derived factor 1a(SDF1a), which acts to retain CXCR4þ BMDCs within tumors (Du et al., 2008; Grunewald et al., 2006; Hattori et al., 2003). Evidence that microenvironmental influences such as low oxygen tension can trigger BMDC recruitment stems from observations in experimentally induced ischemic tissues in which endothelial progenitors and other CXCR4þ BMDCs were recruited, in part through increases in hypoxia-inducible factor 1a (HIF-1a) and its downstream targets SDF1a and VEGF (Ceradini et al., 2004; De Falco et al., 2004; Petit et al., 2007). In another line of investigation, HIF-1a–induced recruitment of proangiogenic BMD CD45þ myeloid cells, which included proangiogenic Tie2þ, VEGFR1þ, CD11bþ, and F4/80þ subpopulations, as well as endothelial and pericyte progenitor cells, was found to promote neovascularization in glioblastoma, a tumor type characterized by extensive hypoxia and necrosis (Aghi et al., 2006; Du et al., 2008). These data suggest that all of these cell types can participate as functionally significant constituents in neovascularization, although MMP-9 expressing monocytic BMDCs appeared to be sufficient to initiate angiogenesis in these tumors (Du et al., 2008). The significant variation in recruitment and utilization of BMDCs in different tumor models underscores the need to further delineate the mechanisms by which they are activated and recruited, as well as their contributions to tumor neovascularization in these contexts. In particular, it will be critical to dissect in more detail the specific BMDC populations recruited into various tumors and better characterize their functional roles in promoting angiogenesis. In this article, we discuss general methods used to visualize and identify recruited BMDCs in two tumor models. We describe the use of bone marrow transplantation to facilitate the analysis of BMDCs, and also present methods that allow the investigator to begin characterizing and isolating the different subtypes of BMDCs found in tumors.

2. Methods for the Visualization of BMDCs in Tumors Adult bone marrow constitutes a reservoir of various stem and progenitor cells capable of contributing to the regeneration of a variety of tissues. Methods whereby genetically marked bone marrow is transplanted

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into tumor-bearing recipient mice have allowed for the positive identification and visualization of BMDCs within tumors. Not only do such approaches permit the tracking and quantification of BMDC recruitment in various tumor systems, they further allow for the isolation and functional characterization of the various BMDC populations in promoting tumor vessel growth. Bone marrow transplantation of mouse tumor models also offers the opportunity to study the angiogenic roles of the various BMDC subtypes in the context of genetic and pharmaceutical manipulations, as well as the mechanisms by which they are recruited to tumors. As more is understood about how BMDCs contribute to tumor neovascularization, it will be important to continue refining the identity of these cells and characterizing their functions.

2.1. Transplantation of GFP-expressing bone marrow to visualize BMDC in tumors Bone marrow transplantation techniques have been widely used in animal models to study a broad spectrum of biological phenomena. Genetic marking of bone marrow unique to that of the donor animal allows one to detect the specific presence of BMDCs within tissues of recipient animals. One example of this approach is to transplant bone marrow from male donor mice into female recipients (Ahn and Brown, 2008). The presence of cells originating from bone marrow can subsequently be detected by in situ hybridization with a Y-chromosome probe. In the following methods sections, we will use GFP-expressing bone marrow cells from b-actineGFP donor mice to fluorescently mark BMDCs transplanted into nonfluorescent recipient animals. This approach provides several advantages: (1) it allows for analysis of bone marrow reconstitution at any time by simply taking a peripheral blood sample and analyzing by flow cytometry; (2) no further staining of the BMDCs is necessary when performing histologic or immunofluorescent analyses; and (3) it permits the detection, fractionation, quantification, and marker characterization of tissue-incorporated BMDCs by flow cytometry. One of most important considerations when optimizing a bone marrow transplantation protocol is determination of the lethal irradiation dose. As different strains of mice tolerate varying levels of irradiation, it is a good idea to perform an initial kill curve to avoid overly lethal doses, or conversely, sublethal doses that result in chimeric bone marrow reconstitution. We routinely apply these methods to two mouse tumor models: C57BL6 b-actin EGFP donor mice with C57BL6 Rip1Tag2 recipient mice (pancreatic islet cell carcinoma) (Song et al., 2005), and FVBN b-actin-EGFP Rag1ko donor mice with FVBN Rag1ko recipient mice (brain tumor model in which glioblastoma cells are orthotopically injected (Du et al., 2008).

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2.1.1. Bone marrow transplantation Reagents C57BL6 b-actin EGFP donor mice C57BL6 Rip1Tag2 recipient mice FVBN b-actin-EGFP Rag1ko donor mice FVBN Rag1ko recipient mice Irradiator Antibiotic water: 1.1 g/l Neomycin sulfate (Sigma-Aldrich, St. Louis, MO, N-1876), 106 units/l polymyxin B sulfate (Sigma-Aldrich, P-1004) final concentration in water, filter sterilized Sterile phosphate-buffered saline lacking Ca2þ and Mg2þ (PBS) supplemented with 2% fetal bovine serum (Hyclone, Logan, UT) (PBS-FBS) 5-ml syringe (Becton Dickinson, Franklin Lakes, NJ) 25-gauge, 5/800 needle (Becton Dickinson) 70-mm nylon cell strainer (BD Biosciences, San Jose, CA, 352350) Ficoll density-gradient solution (density, 1.119) (Histopaque-1119, SigmaAldrich, 11191) Hemacytometer {1/2} cc, 28-gauge insulin syringes (Becton Dickinson) Methods

1. Prepare and give antibiotic water to recipient mice the day before irradiation. 2. Lethally irradiate recipient mice the day before bone marrow transfer. For initial studies, perform a killing curve to establish the lethal dose: Irradiate four mice per group at 800 R, 900 R, 1000 R, and 1200 R. Use the minimum dose at which all four animals in a group die 12 to 15 days after irradiation. The total lethal dose should be split into two equal half-doses 3 h apart. If animals die within 2 to 3 days after irradiation, the mice are likely suffering from gastrointestinal toxicity and the dose is too high. If the mice do not die after 15 days, then the dose is likely sublethal. 3. The next day, anesthetize and euthanize GFP donor mice. Using sterilized tools, dissect out femurs and tibias, removing the skin and as much muscle and soft tissue as possible. 4. Place bones in a dish containing sterile PBS on ice, and then move to a tissue culture hood for all remaining work. 5. Using another set of sterile tools, clean bones of any residual muscle or tissue, and then place cleaned bones into another dish containing sterile PBS. 6. Separate the femur from the tibia by cutting at the knee. Cut the ends of the bones to reveal the medullary compartment containing the marrow.

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7. Fill the 5-ml syringe with cold PBS-FBS and attach the 25-gauge needle. Flush out the bone marrow from each bone into a 50-ml conical tube on ice. Flush from both ends of each bone to ensure maximal recovery. 8. Repeat until all bones have been flushed. 9. Place a 70-mm nylon cell strainer onto a new 50-ml conical tube and pipette the flushed bone marrow through to remove debris. Adjust final volume to 20 ml with PBS-FBS if necessary. 10. Gently add to the bottom of the tube an equal volume (20 ml) of prewarmed, room-temperature Histopaque 1119. 11. Centrifuge for 30 min at 700 g, room temperature, with the brake set to off. 12. Pipette the interphase containing the bone marrow cells into a new 50-ml conical tube. Adjust the final volume to 50 ml with PBS-FBS. 13. Centrifuge for 8 min at 1200 rpm, 4  C. 14. Resuspend the pellet in 25 ml of PBS-FBS and count the cells on a hemacytometer. 15. Centrifuge again for 8 min at 1200 rpm, 4  C, and then resuspend the bone marrow cells at 1 to 2  106 cells/100 ml. 16. Intravenously transfer 1 to 2  106 bone marrow cells (100 ml) to each irradiated recipient mouse using {1/2} cc, 28-gauge insulin syringes.

2.2. Visualization of GFPþ BMDC subpopulations in tumors The investigator can visually verify the recruitment of BMDCs within tumors from GFP-bone marrow transplanted mice by the presence of GFP-positive cells in tumor sections. Quantification and spatial distribution of the GFP-positive BMDCs within tumors in relation to tumor vascular morphology can also be simultaneously analyzed. In addition, the identities of these BMDCs can begin to be determined by immunohistochemical or immunofluorescent staining of lineage specific markers. Here we discuss how tumor-bearing mice are perfused and the relevant tissues prepared for histologic analysis. We will focus on immunofluorescent characterization of the various CD45þ monocytic BMDC subtypes that have been found to considerably modulate tumor neovascularization in an orthotopic model of mouse GBMs (Du et al., 2008). 2.2.1. Cardiac perfusion, tissue fixation, and processing for immunohistochemical analysis Reagents Hemostat 2% 2,2,2-tribromoethanol (Avertin, Sigma-Aldrich) Phosphate-buffered saline lacking Ca2þ and Mg2þ (PBS)

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4% paraformaldehyde in PBS (PFA, Sigma-Aldrich) 25-gauge winged infusion set (Becton Dickinson) 10-ml syringe (Becton Dickinson) 20-ml syringe (Becton Dickinson) Fluorescein-conjugated (FITC) Lycopersicon esculentum lectin (Vector Laboratories, Burlingame, CA, FL-1171), diluted 1:1 with sterile PBS before use Rhodamine Ricinus communis Agglutinin I (Vector RL-1082) 30% sucrose in water, sterile filtered Optimal Cutting Temperature (OCT) Compound (Sakura Tissue-Tek, Torrance, CA, 4583) Dry ice Peel-A-Way tissue embedding molds (Polysciences, Warrington, PA, 18646A) Aluminum foil Cryostat Methods

1. Anesthetize tumor-bearing mouse by intraperitoneal injection of Avertin (250 to 400 mg/kg). 2. Preload 10 ml of PBS into 10-ml syringe and 20 ml of 4% PFA into 20-ml syringe. 3. Immerse tail in warm water to dilate veins in preparation for lectin infusion. 4. Intravenously inject 100 ml of FITC lectin (green fluorescence) or rhodamine agglutinin I (red fluorescence) via tail vein if desired. Wait 3 min for lectin to circulate before cardiac perfusion. 5. Tack mouse arms down with belly side up. Open peritoneal cavity and make an incision along each side of the sternum. Retract the sternum with the hemostat to allow for visualization of the heart. 6. Attach 25-gauge winged infusion set to 10-ml syringe containing PBS, and heart infuse PBS 1 minute. 7. Attach the same 25-gauge winged infusion set to the 20-ml syringe containing 4% PFA, and heart infuse PFA 3 min. Avoid removal of the needle from the heart during syringe transfer. 8. Collect the relevant tissue and place in 10 ml of 4% PFA in a 15-ml conical tube. Cover tube with aluminum foil if tissue contains GFP bone marrow or fluorescent lectin. Place on rocker at 4  C for 6 h. 9. Remove 4% PFA and wash three times with PBS. 10. Immerse tissue in 30% sucrose and place back on rocker at 4  C overnight (12 to 24 h).

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11. Wash tissue three times with PBS. 12. Squeeze a few drops of OCT into an empty tissue-embedding mold and carefully lay tissue in, avoiding air bubbles. Orient according to tumor location or other considerations. 13. Fill cryo-mold with OCT, completely covering the tissue. 14. Cover with aluminum foil and freeze each block on dry ice (10 to 15 min), and then store frozen blocks at –80  C. 15. Cut sections on a cryostat. 2.2.2. Immunohistochemical staining of bone marrow–derived monocytic modulatory cells in tumor sections Reagents PAP PEN hydrophobic barrier pen (The Binding Site, San Diego, AD100) Phosphate buffered saline, Ca2þ and Mg2þ free (PBS-CMF) Blocking buffer: 5% normal goat serum (NGS, Jackson Immunoresearch Lab, West Grove, PA) in PBS-CMF Staining buffer: 2% normal goat serum, 0.3% Triton-X-100 (Sigma-Aldrich) in PBS-CMF PBD wash buffer: 0.1% Tween-20 (Sigma-Aldrich) in PBS-CMF Humidified chamber Rat IgG2b anti-mouse CD45 antibody (BD Biosciences 550539, Clone 30-F11) Rat IgG2b anti-mouse VEGFR1 antibody (Imclone Systems, Clone MF1) Rat IgG1 anti-mouse Tie-2 antibody (eBioscience, San Diego, CA, 145987, Clone TEK4) Rat IgG2b anti-mouse CD11b antibody (BD Biosciences 550282, Clone M1/70) Rat IgG2b anti-mouse F4/80 antibody (Serotec, Oxford, UK, MCAP497, Clone C1:A3-1) Rat IgG2a anti-mouse CD31 antibody (BD Biosciences 553370, Clone MEC 13.3) Rat IgG isotype-matched control antibodies Alexa Fluor 594 goat anti-rat IgG (HþL) secondary detection antibody (Invitrogen-Molecular Probes, Eugene, OR, A-11007) or other desired fluorophore ProLong Gold antifade reagent with DAPI (Invitrogen-Molecular Probes, P-36931) Coverslips Clear nail polish 4% paraformaldehyde (PFA) in PBS Fluorescence microscope (Zeiss Axiophot, Carl Zeiss, Germany)

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Methods

1. Wash sections 3 times, 5 minutes per wash, in PBS-CMF. 2. Draw circles around tissue on the slide with the PAP PEN to create a hydrophobic barrier. Pipette subsequent solutions within this ring. 3. Block in blocking buffer for 30 min at room temperature. 4. Incubate with the desired primary antibody (rat anti-mouse CD45, VEGFR1, Tie-2, CD11b, or F4/80) in staining buffer at 4  C overnight in a humidified chamber. 5. Use rat IgG isotype control antibodies as negative controls on duplicate sections. 6. Wash with PBD 3 times, 5 minutes per wash. 7. Wash with PBS-CMF for 5 min. 8. Incubate with secondary Alexa Fluor 594-conjugated goat antirat antibody (1:200 dilution) in staining buffer for 1 h at room temperature in a humidified chamber. 9. Wash with PBD twice, 5 minutes per wash. 10. Wash with PBS-CMF for 5 min. 11. Mount sections with one or two drops of Prolong Gold antifade reagent with DAPI. Cover with a coverslip, to avoid trapping air bubbles. 12. Cure for 24 h at room temperature in the dark. 13. Seal edges with clear nail polish. 14. Analyze with a fluorescent microscope and store at 4  C short term or – 20  C or colder long term. 15. BMD monocytic modulatory cells can be identified by positive staining of the described markers along with GFP positivity if animals have been transplanted with GFP bone marrow. Fig. 3.1A shows an example of GFPþ BMDCs found within an orthotopic glioblastoma, and Fig. 3.1B reveals CD11b staining of GFPþ BMDCs. Immunohistochemical staining of vasculature 1. Postfix sections in 4% PFA for 20 min at 4  C, protected from light. 2. Wash sections 3  5 min in PBS-CMF. 3. Draw circles around tissue on the slide with the PAP PEN to create a hydrophobic barrier. Pipette subsequent solutions within this ring. 4. Block in blocking buffer for 30 min at room temperature. 5. Incubate with rat anti-mouse CD31 antibody (1:100 dilution) in staining buffer at room temperature overnight in a humidified chamber. 6. Use rat IgG isotype control antibodies as a negative control on duplicate sections. 7. Wash with PBD 3  5 min. 8. Wash with PBS-CMF for 5 min. 9. Incubate with secondary Alexa Fluor 594 (or other desire fluorophore)– conjugated goat antirat antibody (1:200 dilution) in staining buffer for 1 h at room temperature in a humidified chamber.

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A

B BM-GFP-cells

DAPI

BM-GFP-cells

CD11b

C BM-GFP-cells CD31 DAPI

Figure 3.1 Visualization and immunohistochemical staining of BMDCs in tumors. Orthotopic glioblastomas implanted into GFP-bone marrow transplanted mice were sectioned, stained, and analyzed by fluorescence microscopy. (A) GFPþ bone marrow cells (green) are readily detected within the tumor. Scale bar, 50 mm. (B) Staining of tumor sections identifies a fraction of GFPþ bone marrow cells (green) recruited to the tumor as CD11bþ myeloid cells (red and green merge). Scale bar,15 mm. (C) Staining of the tumor vasculature with CD31antibodies (red) reveals dilated tumor vessel morphology, while recruited GFPþ bone marrow cells (green) reside among tumor cells (yellow arrowheads) and are also incorporated into the vasculature (white arrowheads). Scale bar,15 mm.

10. Wash with PBD 2  5 min. 11. Wash with PBS-CMF for 5 min. 12. Mount sections with one or two drops of Prolong Gold antifade reagent with DAPI. Cover with a coverslip, and avoid trapping air bubbles. 13. Cure for 24 h at room temperature in the dark. 14. Seal edges with clear nail polish. 15. Analyze with fluorescent microscope and store at 4  C short-term or – 20  C or colder long term. 16. Vasculature can be visualized without immunohistochemical staining if tissues were perfused with fluorescent lectin prior to animal euthanization. CD31 staining enhances and allows for complete staining of the vasculature regardless of vessel functionality. Fig. 3.1C shows staining of tumor vasculature with CD31, revealing hyperdilated vessels alongside GFPþ BMDCs.

2.3. Quantification of GFPþ BMDC subpopulations in tumors While tumor-recruited BMDCs can be quantified by counting the number of GFP-positive cells (and relevant marker stained cells) in tissue sections, a more efficient and versatile way to quantify and characterize BMDCs is by flow cytometry. FACS analysis allows for GFP-positive BMDCs to be quickly identified and quantified, and further fractionation of BMDC

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subtypes can be easily performed by staining with the relevant markers. In the following we discuss the methods used to analyze BMDCs in tumor tissues by flow cytometry, specifically detecting for endothelial progenitor cells, pericyte progenitor cells, and various CD45þ monocytic modulatory cells (Du et al., 2008). 2.3.1. Flow cytometric analysis of bone marrow–derived vascular progenitor and modulatory cells in tumors Reagents 2% 2,2,2-tribromoethanol (Avertin, Sigma-Aldrich) Collagenase digestion buffer: 0.2 g bovine serum albumin (BSA, Sigma Aldrich, B4287) 12,500 units collagenase II (Worthington Biochemical, Lakewood, NJ, LS004176) 12,500 units collagenase IV (Worthington Biochemical, LS004188) 20 ml DNase I (RNase, Proteinase-Free, Worthington Biochemical, LS006333) 20 ml PBS 70-mm nylon cell strainers (BD Biosciences 352350) 5-ml syringes BD Pharm Lyse 10, red-blood-cell lysing buffer (BD Biosciences 555899) Phosphate-buffered saline lacking Ca2þ and Mg2þ supplemented with 5% fetal bovine serum (Hyclone SH30070) (5% FBS-PBS) Phosphate-buffered saline lacking Ca2þ and Mg2þ supplemented with 2% fetal bovine serum (Hyclone SH30070) (2% FBS-PBS) Fc Block: rat anti-mouse CD16/CD32 (BD Biosciences 553142) Phycoerythrin (PE)-labeled, isotype-matched control Allophycocyanin (APC)-labeled isotype-matched control PE-Cy7–labeled, isotype-matched control Alexa Fluor 647–labeled, isotype-matched control Antibodies for detection of monocytic modulatory cells: PE-Cy7-conjugated rat IgG2b anti-mouse CD45 antibody (eBioscience 25-0451, clone 30-F11) APC-conjugated rat IgG2b anti-mouse CXCR4 antibody (BD Biosciences 558644, clone 2B11) PE-conjugated rat IgG1 anti-mouse Tie-2 antibody (eBioscience 12-5987, clone TEK4) PE-conjugated rat IgG2a anti-mouse F4/80 antibody (eBioscience 12-4801, clone BM8) PE-conjugated rat IgG2b anti-mouse CD11b antibody (eBioscience 12-0112, clone M1/70)

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Biotinylated rat IgG2b anti-VEGFR1 antibody clone MF1 (Imclone Systems), biotinylation of antibody must be performed (FluoReporter Mini-biotin-XX Protein Labeling Kit, Invitrogen-Molecular Probes F6347) Streptavidin-PE (eBioscience 12-4317) Antibodies for detection of endothelial progenitor cells: PE-conjugated rat IgG2a anti-mouse VEGFR2 antibody (eBioscience 12-5821, clone Avas12a1) Alexa 647-conjugated rat IgG1 anti-mouse VE-cadherin antibody (eBioscience 51-1441, clone BV13) Antibodies for detection of pericyte progenitor cells: PE-conjugated rat IgG2a anti-mouse PDGFRb antibody (eBioscience 12-1402, clone APB5) APC-conjugated rat IgG2a anti-mouse Sca-1 antibody (eBioscience 17-5981, clone D7) BD Via-Probe cell viability solution (ready-to-use 7-AAD solution, BD Biosciences 555815) BD LSR II flow cytometer (BD Biosciences) 5-ml polystyrene round-bottom tubes (Becton Dickinson) Methods

1. Anesthetize bone marrow–transplanted, tumor-bearing mice by intraperitoneal injection of Avertin (250 to 400 mg/kg). 2. Carefully dissect out tumor and place in a dish containing PBS on ice. 3. Finely mince tumor with a razor blade on ice in a clean and dry dish. 4. Transfer minced tumor into 10 ml of collagenase digestion buffer in a 50-ml conical tube. 5. Incubate in a 37  C water bath for 13 min, with manual shaking/ stirring every 2 to 3 min. 6. Immediately place on ice and stop digestion by adding 5% FBS-PBS up to 40 ml. 7. Strain cells through a 70-mm nylon cell strainer into a new 50-ml conical tube. 8. Remove the plunger from a 5-ml syringe and vigorously rub and scrape any residual chunks of tumor tissue on top of the strainer against the mesh. Rinse with 10 ml of 5% FBS-PBS to maximize tumor cell recovery. 9. Centrifuge at 1200 rpm for 5 min at 4  C. 10. Aspirate supernatant and resuspend cell pellet in 1 ml of 1 Pharm Lyse (diluted in water). Place on ice for 3 min, and then stop reaction by adding 5% FBS-PBS to 50 ml.

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11. Centrifuge at 1200 rpm for 5 min at 4  C. 12. Resuspend cells in 2% FBS-PBS according to the cell yield and the number of controls and staining combinations required; 0.5 to 1  106 cells per 100 ml are recommended. Split the cells in 100-ml aliquots into 5-ml polystyrene, round-bottom tubes. 13. Set aside single color- and isotype-matched control samples. Add Fc block to remaining samples at 1:50 dilution. 14. Apply primary antibody multicolor combinations according to Table 3.1 to detect different populations of BMD CD45þ monocytes, endothelial progenitor cells, or pericyte progenitor cells. The FITC channel is reserved for identifying GFP-positive BMDCs in the tumor sample, and 7-AAD is reserved for staining and exclusion of dead cells. 15. Apply appropriate antibodies to single color controls and isotypematched controls. 16. Incubate primary antibody combinations on ice for 30 min, protected from light. 17. Wash cells with 3 ml of 2% FBS-PBS, and centrifuge at 1200 rpm for 5 min at 4  C. 18. Resuspend cells in 200 ml of 2% FBS-PBS, except for staining of VEGFR1þ hemangiocytes. 19. For detection of VEGFR1þ hemangiocytes, resuspend cells in 400 ml of 2% FBS-PBS and add streptavidin-PE at a 1:400 dilution. Incubate on ice for 30 min, protected from light. Following secondary staining, wash and centrifuge as described in Step 19. Resuspend in 200 ml of 2% FBS-PBS. 20. Prior to analysis of each sample, add 20 ml of BD Via-Probe cell viability solution (7-AAD) to each tube and incubate for 10 min. Analyze samples on the flow cytometer. 21. Exclude dead cells and gate for GFPþ BMDCs. Use single-color and isotype-matched controls to set gates for marker analysis. Fig. 3.2 demonstrates the identification and analysis of GFPþ CD45þ CD11bþ BMDCs in an orthotopic glioblastoma tumor by FACS analysis.

3. Analysis of Pericyte Progenitors in Tumors RIP1Tag2 mice express the viral SV40Tag oncoproteins under the control of the rat insulin gene II promoter in the pancreatic b cells of the 400 islets of Langerhans, setting in motion a multistep pathway to beta islet cell carcinoma as characterized by the temporal and synchronistic appearance of distinctive lesional stages in all transgenic mice (Bergers et al., 1998; Hanahan, 1985). Aberrant hyperproliferation of the b cells starts at about 3 to 4 weeks of age, producing hyperplastic and dysplastic

Table 3.1 Antibody and fluorochrome combinations for detection and analysis of BMDCs by flow cytometry.

Color

TEMs

FITC PE PE-Cy7 APC 7-AAD

GFP-BM Tie-2 CD45 CXCR4 Dead cells

Pericyte progenitor cells

Monocytic accessory modulatory cells

Endothelial progenitor cells

Tumor- associated macrophages

Myeloid cells

Hemangiocytes

Color

GFP-BM F4/80 CD45 CXCR4 Dead cells

GFP-BM CD11b CD45 CXCR4 Dead cells

GFP-BM VEGFR1 B* CD45 CXCR4 Dead cells

FITC PE

GFP-BM VEGFR2

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Figure 3.2 Flow cytometric analysis and characterization of BMDCs in tumors. Orthotopic glioblastomas implanted into GFP-bone marrow transplanted mice were resected and processed into a single cell suspension for flow cytometric analysis. In this example, the tumor cell suspension was stained with PE-Cy7 conjugated CD45 antibodies and PE conjugated CD11b antibodies to detect and quantify BMD myeloid cells, and then counterstained with 7-AAD. During anaylsis, cellular debris and 7-AADþ dead cells were sequentially gated out, followed by selective gating of GFPþ cells within the tumor suspension for interrogation of CD45 and CD11b expression. Gating parameters for positive CD45 and CD11b expression were set by staining bone marrow cells with isotype-matched control antibodies or CD45 and CD11b antibodies alone (lower left corner). Note that the majority of GFPþ BMDCs recruited in this tumor model are CD45þ monocytes, of which about one-fourth of them are CD11bþ myeloid cells.

islets, initially with a quiescent vasculature. Then, at 6 to 7 weeks, angiogenesis is switched on in a subset of the dysplastic islets. Subsequently, solid tumors form at about 10 to 11 weeks as encapsulated tumors and invasive carcinomas. The mice die between 13.5 to 15 weeks of age with substantial tumor burden and consequent hyperinsulinemia. Tumor pericytes originate not only from a pre-existing pool of pericytes, but also by maturation of undifferentiated progenitors recruited to the

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newly formed tumor blood vessels. In pancreatic islet tumors of Rip1Tag2 mice, PDGFRb is expressed in perivascular cells closely associated with the tumor vasculature, while the PDGF ligands B and D for PDGFRb are expressed in tumor endothelial cells, reflective of paracrine communication pathways similar to the vascular processes during development (Bergers et al., 2003; Hellstrom et al., 1999; Song et al., 2005). One advantage of studying pericyte progenitors in pancreatic islet tumors is based on the fact that PDGFRb is exclusively expressed in pericytes in these tumors and that these tumors are rather devoid of stromal fibroblasts that can also express PDGFRb. We have identified PDGFRbþ pericyte progenitors (PPPs) closely associated with angiogenic endothelial cells in Rip1Tag2 tumors. Furthermore, a subset of these cells is recruited from the bone marrow, which indicates that recruitment of BMD cells to sites of a growing vasculature is not limited to endothelial cells, but can also include pericytes (Song et al., 2005). Importantly, three populations of tumor pericytes can be detected in tumors, each reflecting a distinct differentiation stage: (1) PPPs that are positive for PDGFRb but negative for the mature pericyte markers NG2, desmin, and a-SMA; (2) intermediate pericytes that express PDGFRb and the mature pericyte markers NG2, desmin, and a-SMA; and (3) mature pericytes that are positive for the mature pericyte markers NG2, desmin, and a-SMA, but have lost the expression of PDGFRb. The three populations of tumor pericytes can be detected by FACS and immunohistochemistry of tumor sections. Pericyte progenitors can also be isolated from tumor cell suspensions using magnetic beads based on PDGFRb expression. They can later be cultured as monoculture in vitro to allow their differentiation into mature pericytes. However, we have demonstrated that in order to upregulate desmin expression, it is necessary to co-culture pericyte progenitors with endothelial cells. We use the 3D Matrigel gel as an in vitro assay, which permits the investigator to observe the interactions between endothelial cells and pericyte progenitors during vessel formation.

3.1. Detection of pericyte progenitors and pericytes in tumors Prior to sacrifice, mice are injected intravenously with FITC-conjugated tomato lectin to detect the blood vessels, and tumor tissues are fixed and processed as described in Section 2.2.1. Tumor pericytes are detected by immunostaining of 16-mm frozen sections with antibodies against PDGFRb, NG2, desmin, and a-SMA. Commercially available desmin and a-SMA antibodies are of the mouse isotype. Hence, to avoid unspecific background, antibodies are conjugated to the AlexaFluor647 fluorochrome just prior to immunostaining using the Zenon AlexaFluor647 Mouse IgG-specific Labeling Kit (Invitrogen), according to the manufacturer’s instructions.

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Reagents

PAP PEN hydrophobic barrier pen (The Binding Site) Phosphate buffered saline, Ca2þ and Mg2þ free (PBS) Fixation buffer: 4% paraformaldehyde (PFA) in PBS Blocking/permeabilization buffer: PBS supplemented with 5% normal goat serum (NGS; Jackson Immunoresearch Lab) and 0.3% Triton X-100 (Sigma-Aldrich) Incubation buffer: PBS supplemented with 2% NGS and 0.3% Triton X-100 Washing buffer: PBS supplemented with 0.1% Tween-20 (Sigma-Aldrich) Zenon AlexaFluor647 Mouse IgG1 Labeling Kit (Invitrogen) Zenon AlexaFluor647 Mouse IgG2a Labeling Kit (Invitrogen) Rabbit polyclonal anti-NG2 chondroitin sulfate proteoglycan antibody (Chemicon, Temecula, CA), used at 2 mg/ml Mouse IgG1 monoclonal anti-desmin antibody (Clone D33, DAKO, Carpinteria, CA), used at 1:200 Mouse IgG2a monoclonal anti-alpha smooth muscle actin (a-SMA) antibody (Clone 1A4, DAKO), used at 1:500 Rat IgG2a monoclonal, anti-PDGFRb antibody (Clone APB5, eBiosciences), used at 10 mg/ml Rabbit IgG isotype-matched control ( Jackson Immunoresearch Lab) Mouse IgG1 and IgG2a isotype-matched controls (BD Biosciences) Rat IgG2a isotype-matched control (eBiosciences) AlexaFluor647-conjugated goat anti-rabbit IgG (HþL) antibody (Invitrogen-Molecular Probes), used at 1:400 AlexaFluor546-conjugated goat anti-rat IgG (HþL) antibody (InvitrogenMolecular Probes), use at 1:400 ProLong Gold antifade reagent with DAPI nucleic acid stain (InvitrogenMolecular Probes) Fluorescence confocal microscope (Zeiss LSM510, Carl Zeiss, Germany) Methods

1. Draw circles around tissue on the slide with the PAP PEN to create a hydrophobic barrier. Pipette subsequent solutions within this ring. 2. Post-fix the frozen sections in 4% PFA/PBS for 10 min at room temperature. 3. Wash the tissue sections three times in PBS for 5 min at room temperature. 4. Block the tissues with 5%NGS/0.3% Triton X-100/PBS for 30 min at room temperature. 5. Wash the tissue sections once in PBS for 5 min at room temperature. 6. Incubate the sections with the primary antibodies diluted in the incubation buffer overnight at 4  C. Use isotype-matched control antibodies as a negative control on duplicate sections.

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7. Wash the tissue sections twice in PBS/0.1% Tween-20 for 5 min at room temperature. 8. Wash the tissue sections in PBS for 5 min at room temperature. 9. Incubate the sections with the secondary antibodies diluted in the incubation buffer for 1 h at room temperature. 10. Wash the tissue sections twice in PBS/0.1% Tween-20 for 5 min at room temperature. 11. Wash the tissue sections in PBS for 5 min at room temperature. 12. Mount the slides with ProLong Gold antifade reagent with DAPI nucleic acid stain included. 13. Evaluate immunostaining with a fluorescence confocal microscope. Fig. 3.3 demonstrates pericyte staining of a Rip1Tag2 tumor with desmin and PEGFRb. 14. Isolation of pericyte progenitors and pericytes in tumors. 15. PDGFRbþ pericyte progenitors are isolated from single-cell suspensions of Rip1Tag2 tumors. Given that this progenitor population represents a small percentage of the total tumor cell population (2%), it is necessary to start with a high number of cells, that is, five to seven mice per isolation. 3.1.1. Preparation of single-cell suspension from Rip1Tag2 tumors Reagents Sterile conical 50-ml tubes Sterile Petri dishes One disposable 70-mm cell strainer (Becton Dickinson) One sterile razor blade Lectin

Desmin

Lectin PDGFRβ

Merge

Figure 3.3 Visualization of tumor pericytes on tissue sections.13.5-week-old Rip1Tag2 mice were injected intravenously with FITC-conjugated tomato lectin that binds to blood vessels and allows their visualization in pancreatic tumors (green).Tumor sections are then stained with antibodies against PDGFRb (red) and desmin (blue). Pictures were acquired using a fluorescent confocal microscope. Scale bar, 50 mm.

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Water bath (37  C) 2% 2,2,2-tribromoethanol (Avertin, Sigma-Aldrich) Sterile phosphate-buffered saline without Ca2þ and Mg2þ (PBS) Sterile PBS supplemented with 63.7 mg/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml fungizone Collagenase digestion buffer: 0.2 g bovine serum albumin (BSA, Sigma Aldrich) 12,500 units collagenase II (Worthington Biochemical) 12,500 units collagenase IV (Worthington Biochemical) 20 ml DNase I (RNase, proteinase-free, Worthington Biochemical) 20 ml PBS Phosphate-buffered saline lacking Ca2þ and Mg2þ supplemented with 5% fetal bovine serum (Hyclone) (5% FBS-PBS) Phosphate-buffered saline lacking Ca2þ and Mg2þ supplemented with 2% fetal bovine serum (Hyclone) and 1 mM EDTA (Sigma-Aldrich) (2% FBS-PBS-EDTA) Ficoll density gradient solution (density 1.119) (Histopaque-1119, SigmaAldrich) 0.4% trypan blue solution (Sigma-Aldrich) Methods

1. Euthanize 13-week-old Rip1Tag2 mice bearing substantial tumor burden by intraperitoneal injection of Avertin (250 to 400 mg/kg). 2. Harvest the pancreases and wash them in PBS containing antibiotics and fungizone and place them in fresh solution on ice. 3. Carefully dissect the tumors out of the pancreas on ice and place them in fresh PBS containing antibiotics and fungizone. 4. Place the tumors in a clean, sterile Petri dish, and finely mince them with a sterile razor blade. Add the minced tumors to 20 ml of collagenase digestion buffer. 5. Incubate in a 37  C water bath for 13 min, with manual shaking/ stirring every 2 to 3 min. 6. Add 20 ml of 5% FBS-PBS and mechanically dissociate the digested tissue by pipetting up and down. 7. Place a 70-mm disposable cell strainer on top of a 50-ml conical tube and filter the digested tumor solution. 8. Remove the plunger from a 5-ml syringe and vigorously rub and scrape any residual chunks of tumor tissue on top of the strainer against the mesh. Rinse with 10 ml of 5% FBS-PBS to maximize tumor cell recovery. 9. Centrifuge at 1200 rpm for 8 min at 4  C. 10. Resuspend the cell pellet into 10 ml of 2% FBS-PBS-EDTA at room temperature. 11. Transfer 10 ml of Histopaque-1119 into a 50-ml conical tube.

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12. Carefully layer 10 ml of tumor cell suspension onto the Histopaque1119 layer 13. Centrifuge at 700  g for 30 min at room temperature with the brake set to off. 14. Red blood cells pellet at the bottom of the tube while cells from the tumors band at the interface between the Histopaque-1119 and the upper layer. Carefully aspirate the cells with a Pasteur pipette without disturbing the interface and transfer into a 50-ml conical tube. 15. Bring the cell suspension to a total volume of 50 ml by adding 2% FBSPBS-EDTA. Transfer 10 ml into an Eppendorf tube for cell counting. Centrifuge the rest at 1200 rpm for 8 min at 4  C. 16. Cell counting: Mix 10 ml of tumor cell suspension and 10 ml of trypan blue (final dilution 1:2). Determine the total number of cells using a hemacytometer chamber.

3.2. Isolation of PDGFRbþ pericytes from Rip1Tag2 tumor cell suspension Reagents

One sterile, polystyrene 5-ml, round-bottom tube (Becton Dickinson) Phosphate-buffered saline lacking Ca2þ and Mg2þ supplemented with 2% fetal bovine serum (Hyclone) and 1 mM EDTA (Sigma-Aldrich) (2% FBS-PBS-EDTA) EasySep magnet (cat. 18000, Stem Cell Technologies, Canada) EasySep Biotin Selection Kit (cat. 18556, Stem Cell Technologies). Contains the mouse-specific Fc Receptor blocking antibody, the EasySep Biotin Selection Cocktail and EasySep Magnetic Nanoparticles Biotin-conjugated Rat IgG2a anti-PDGFRb antibody (Clone APB5, eBiosciences), used at 7.5 mg/ml Methods

1. PDGFRbþ pericyte progenitors are isolated from Rip1Tag2 mouse tumors using the EasySep Biotin Selection Kit according to the exact manufacturer’s instructions, as described below. 2. Resuspend the cell pellet at 2  108 cells/ml in 2% FBS-PBS-EDTA and place the cell suspension into a 5-ml, round-bottom polystyrene tube. 3. Add mouse-specific Fc receptor blocking antibody at 10 ml/ml. Mix well. 4. Add biotin-conjugated rat anti-PDGFRb antibody. Mix well and incubate at room temperature for 15 min. 5. Add EasySep Biotin Selection Cocktail at 100 ml/ml. Mix well and incubate at room temperature for 15 min. 6. Add EasySep Magnetic Nanoparticles at 50 ml/ml. Mix well and incubate at room temperature for 10 min.

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7. Bring the cell suspension to a total volume of 2.5 ml by adding 2% FBSPBS-EDTA. Mix the cells in the tube by gently pipetting up and down. Place the tube (without cap) into the magnet. Set aside for 5 min. 8. Pick up the magnet, and in one continuous motion invert the magnet and tube, pouring off the supernatant fraction. The magnetically labeled cells remain inside the tube. Leave the magnet and tube in inverted position for 2 to 3 s, and then return to upright position. 9. Remove the tube from the magnet and add 2.5 ml 2% FBS-PBSEDTA. Mix the cells by gently pipetting up and down. Place the tube back in the magnet and set aside for 5 min. 10. Repeat Steps 7 and 8 twice, and then Step 7 once more, for a total of four separations in the magnet. Remove the tube from the magnet and resuspend cells in differentiation medium.

3.3. In vitro differentiation of pericyte progenitors PDGFRbþ pericyte progenitors isolated from pancreatic Rip1Tag2 tumors have the capacity to differentiate into mature pericytes in vitro (Song et al., 2005). Here we describe procedures to differentiate pericyte progenitors both under monoculture conditions, as well as co-cultures with endothelial cells on a 3D Matrigel matrix. 3.3.1. In vitro differentiation of pericyte progenitors as monoculture Reagents Eight-well Labtek Permanox chamber slides (Nalge Nunc Int., Rochester, NY) Coating solution: PBS supplemented with 0.1% gelatin from porcine skin, type A (Sigma-Aldrich) Differentiation medium: mesenchymal stem cell basal medium (MSCBM, Lonza Group, Switzerland) supplemented with MSCBM SingleQuot Kit (Lonza Group) and 5 ng/ml of recombinant human TGFb1 (R&D Systems, Minneapolis, MN). Methods

1. Incubate an eight-well Labtek Permanox slide chamber with 0.1% gelatin/PBS for 1 h at 37  C. 2. Remove the uncoated 0.1% gelatin/PBS. Wash once with PBS. 3. Transfer freshly isolated PDGFRbþ pericyte progenitors to gelatincoated slide chamber at a density of 10,000 cells per well in differentiation medium.

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4. Culture the cells for 7 days at 37  C in 5% CO2. Replace half the medium every second day. 3.3.2. Differentiation culture of PDGFRbþ pericyte progenitors with endothelial co-cultures in 3D Matrigel Pericytes and endothelial cells are known to closely interact in vivo through both direct contact as well as paracrine signaling mechanisms. Therefore, co-cultures of pericyte progenitors with endothelial cells in vitro may more closely mimic the maturation and behavior of pericytes in vivo. Indeed, we have observed that desmin is upregulated in mature pericytes only under these co-culture conditions. Reagents

Four-well Labtek Permanox chamber slides (Nalge Nunc Int.) CDC/EU.HMEC-1 (HMEC-1) immortalized human microvascular endothelial cells (Ades et al., 1992) PDGFRbþ pericyte progenitor cells (as isolated in Section 3.2) CellTracker fluorescent cell-labeling probes in color of choice (Molecular Probes-Invitrogen) Endothelial growth medium: MCDB131 (Gibco-Invitrogen) supplemented with 10% FBS, 1 L-glutamine Serum-free MCDB131 medium (no additives) Endothelial Cell Medium-2 (EGM-2, Lonza Group) supplemented with EGM-2 SingleQuot Kit, 63.7 mg/ml penicillin, 100 mg/ml streptomycin, 1 antibiotic antimycotic stabilized solution (AASS, Sigma-Aldrich) Pericyte differentiation medium: mesenchymal stem cell basal medium (MSCBM, Lonza Group) supplemented with MSCBM SingleQuot Kit and 5 ng/ml of recombinant human TGFb1 (R&D Systems) Cold sterile PBS PBS supplemented with 0.5 mM EDTA (Sigma-Aldrich) (PBS-EDTA) Growth-factor-reduced (GFR)-Matrigel (Becton Dickinson) Hemacytometer Chilled sterile pipette tips 4% paraformaldehyde in PBS 0.1 M glycine Methods

1. Culture HMEC-1 immortalized human microvascular endothelial cells in complete MCDB131 endothelial culture medium. 2. Thaw GFR-Matrigel at 4  C on ice overnight.

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3. Under sterile conditions in the tissue culture hood, place GFR-Matrigel and chamber slides on ice. Using chilled pipette tips, mix Matrigel to homogeneity. 4. Pipette enough Matrigel to cover the surface of each chamber on the slide and make a thick gel (150 to 200 ml per square centimeter of growth surface area). 5. Place slides and Matrigel in the tissue culture incubator at 37  C for 30 min. After incubation, check to see that Matrigel has set. 6. Wash HMEC-1 cells with cold PBS and detach from the plates by adding 2 ml of PBS-EDTA. Incubate at 37  C for 1-2 min. After cells have lifted off plate, add 10 ml complete growth medium containing 10% FBS. 7. Spin down cells at 1200 rpm for 5 min, and resuspend the pellet in 1 ml of complete growth medium. 8. Label cells with CellTracker fluorescent probes at a 10-mM working concentration and incubate for 15 min at 37  C. 9. Spin endothelial cells down and resuspend in 10 ml serum-free medium. Incubate for 30 min at 37  C. 10. Spin labeled endothelial cells down and resuspend in 5 to 10 ml of complete EGM-2 medium. 11. Count cells with a hemacytometer and seed 50,000 labeled endothelial cells per square centimeter of growth surface on top of the solidified Matrigel. Add enough complete medium if necessary to ensure complete coverage of the well (final volume 0.5 ml for a four-chamber slide). 12. Place chamber slide containing Matrigel matrices and labeled endothelial cells overnight in the tissue culture incubator. Endothelial tube-like structures should form within a few hours. 13. Isolate PDGFRbþ pericyte progenitor cells as described in Section 3.2. (Optional: Fluorescently label PDGFRbþ pericyte progenitor cells with CellTracker in a color different from that used to label endothelial cells.) Resuspend pericyte progenitor cells in complete MSCBM medium. 14. Aspirate medium from chamber slides, being careful not to disturb the Matrigel bed and endothelial tubes. 15. Seed pericyte progenitor cells at one-third the number of endothelial cells seeded (16,666 pericyte progenitor cells per square centimeter of growth surface) on top of the Matrigel bed containing endothelial tubes. 16. Allow co-cultures to incubate for 3 to 7 days in tissue culture incubator. 17. Rinse cultures with PBS and fix by covering the Matrigel/endothelial cell/pericyte co-cultures with 4% paraformaldehyde for 15 min at room temperature. 18. Rinse with 0.1 M glycine 2  5 min.

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19. Cover chambers with OCT and place on dry ice to freeze. 20. Pry frozen Matrigel OCT blocks from the chambers and store wrapped in foil at –70  C until use. 21. Cut sections on a cryostat, then proceed to stain for mature pericyte markers as described below. 3.3.3. Detection of mature pericytes after differentiation After 7 days of culture or endothelial cell co-culture in differentiation medium, PDGFRbþ pericyte progenitors have acquired a mature pericyte phenotype identified by (1) a changed morphology from round to elongated, and (2) expression of the mature pericyte markers a-SMA, desmin, and NG2. Reagents

Fixation buffer: 4% paraformaldehyde (PFA) in PBS Blocking/permeabilization buffer: PBS supplemented with 5% normal goat serum (NGS, Jackson Immunoresearch Lab) and 0.3% Triton X-100 (Sigma-Aldrich) Incubation buffer: PBS supplemented with 2% NGS and 0.3% Triton X-100 Vector M.O.M. (mouse-on-mouse) blocking reagent (Vectors Lab) Rabbit IgG polyclonal anti-NG2 chondroitin sulfate proteoglycan antibody (Chemicon), used at 2 mg/ml Mouse IgG1 monoclonal anti-desmin antibody (Clone D33, DAKO), used at 1:200 Mouse IgG2a monoclonal anti-a-SMA antibody (Clone 1A4, DAKO), used at 1:500 Rat IgG2a monoclonal anti-PDGFRb antibody (Clone APB5, eBiosciences), used at 10 mg/ml Rabbit IgG isotype-matched control (Jackson Immunoresearch Lab) Mouse IgG1 and IgG2a isotype-matched controls (BD Biosciences) Rat IgG2a isotype-matched control (eBiosciences) AlexaFluor594-conjugated goat anti-mouse IgG (HþL) antibody (Invitrogen-Molecular Probes), used at 1:200 AlexaFluor594-conjugated goat anti-rabbit IgG (HþL) antibody (Invitrogen-Molecular Probes), used at 1:200 AlexaFluor488-conjugated goat anti-rat IgG (HþL) antibody (InvitrogenMolecular Probes), used at 1:200 ProLong Gold antifade reagent with DAPI nucleic acid stain (InvitrogenMolecular Probes) Fluorescence microscope (Zeiss Axiophot, Carl Zeiss, Germany)

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DAPI PDGFRb NG2

DAPI PDGFRb a-SMA

DAPI PDGFRb

Desmin

Figure 3.4 PDGFRbþ pericyte progenitors differentiate into mature pericytes in vitro. PDGFRbþ cells isolated from Rip1Tag2 tumors were cultured in vitro in complete MSCBM medium. After 7 days, cells were fixed and immunostained with antibodies against PDGFRb (green) and NG2, desmin, or a-SMA (red, as indicated).

Figure 3.5 Differentiation of PDGFRbþ pericyte progenitors under co-culture conditions with endothelial cells. Endothelial cells were labeled with CellTracker Green dye and cultured on a 3D Matrigel matrix to form endothelial tube-like structures. Pericyte progenitors were co-cultured with the endothelial cells in differentiation medium for 7 days. Staining for the mature pericyte marker NG2 (red) reveals mature pericytes wrapped around the endothelial tubes. Nuclei are counterstained with DAPI (blue).

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Methods

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Wash cultured PDGFRbþ pericyte progenitors twice in PBS. Fix the cells in 4%PFA/PBS for 15 min at room temperature. Wash the cells three times in PBS, 5 min each. If immunostaining with antibodies against a-SMA or desmin, block the cells with Vector M.O.M. diluted in PBS according to the manufacturer’s instructions. Incubate for 30 min at room temperature. Briefly wash the cells with PBS. Block the cells with 5%NGS/0.3% Triton X-100/PBS for 30 min at room temperature. Incubate the cells with the primary antibodies for 1 h at room temperature (or overnight at 4  C). Wash the cells three times in PBS for 5 min at room temperature. Incubate the cells with the secondary antibodies for 1 h at room temperature. Wash the cells three times in PBS for 5 min at room temperature. Mount the slides with ProLong Gold antifade reagent with DAPI nucleic acid stain included. Evaluate the positive cells using a fluorescence microscope. Figs. 3.4 and 3.5 illustrate staining of differentiated mature pericytes under monoculture and endothelial co-culture conditions, respectively.

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