Chapter 15 - Springer Link

Chapter 15 Tracking Inflammation-Induced Mobilization of Mesenchymal Stem Cells Erika L. Spaeth, Shannon Kidd, and Frank C. Marini Abstract The act of migration is similar for many cell types. The migratory mechanism of mesenchymal stem cells (MSC) is not completely elucidated, yet many of the initial studies have been based on current understanding of the leukocyte migration. A normal function of MSC is the ability of the cell to migrate to and repair wounded tissue. This wound healing property of MSC originates with migration towards inflammatory signals produced by the wounded environment [1]. A tumor and its microenvironment are capable of eliciting a similar inflammatory response from the MSC, thus resulting in migration of the MSC towards the tumor microenvironment. We have shown MSC migration both in vitro and in vivo. In this chapter, we elucidate several in vivo methods to study MSC migration and mobilization to the tumor microenvironment. The first model is an exogenous model of MSC migration that can be performed in both xenograft and syngenic systems with in vitro expanded MSC. The second model utilizes transgenic-fluorescentcolored mice to follow endogenous bone marrow components including MSC. The third technique enables us to analyze data from the transgenic model through multispectral imaging. Furthermore, the migratory phenotype of MSC can be exploited for use in tumor-targeted gene delivery therapy has been efficacious in animal model studies and is an anticipated therapeutic device in clinical trials. Key words: MSC, Inflammation, Tumor bone marrow transplant, Transgenic mice

1. Introduction For the multipotent population of mesenchymal stem (stromal) cells (MSC), migration is an innate function. These cells naturally migrate to wounded environments whether it be a cutaneous lesion, infarcted tissue, or tumor. The MSC is a structurally supportive participant in wound closure, but is also able to promote an immune suppressive response to minimize the inflammation and thus promote healing (2–4).

Mikhail G. Kolonin and Paul J. Simmons (eds.), Stem Cell Mobilization: Methods and Protocols, Methods in Molecular Biology, vol. 904, DOI 10.1007/978-1-61779-943-3_15, © Springer Science+Business Media, LLC 2012

173

174

E.L. Spaeth et al.

MSC have been shown to migrate towards several tumor types including ovarian, breast, colon, prostate, and pancreatic carcinomas as well as lung metastases, sarcomas, and gliomas (5–14). Discordant evidence suggests both pro- and anti-tumorigenic effect of the MSC once in the tumor microenvironment (12, 15–18); however, when used as an anticancer agent delivery vehicle, the capabilities of the cell to migrate is maintained to ensure successful delivery of the MSC to the local tumor environment (9–11, 19, 20). Migration can be monitored in vitro using several common methods including the Transwell (Boyden chamber) Assay, the Scratch Assay, and the modified Ouchterlony Assay. In this chapter, we address in vivo migration models (bioluminescence and fluorescence) that will be evaluated using either syngenic or xenograft murine models that enable the investigator to follow the migration of a labeled cell through noninvasive techniques that allow the potential analysis of long-term physiological relevance of the migration without sacrificing the animal. PET/CT and MRI are not covered herein although the methods have been used to follow MSC in animal models (21–23). In addition, we address the use of fluorescently labeled transgenic murine model bone marrow transplant models that address the question of MSC mobility in an enclosed, endogenous system. These techniques will allow investigators to study the movement and localization of endogenous MSC to wounds and injured tissues including tumors, ischemic tissues, bone fractures, and muscle lacerations. Using multispectral imaging techniques, we are able to follow fluorescently expressing cell position within tissue sections to better understand the incorporation and involvement of MSC within injured tissues on a whole body/whole organ/whole tissue scale. While the following chapter addresses only one aspect of an inflamed environment, the tumor, these protocols are easily adapted to enable investigators to pursue many other forms of inflammatory induced migration of MSC.

2. Materials 2.1. MSC Isolation and Culture

1. Alpha Modified Eagle’s Medium (α-MEM) with supplements (L-glutamine and penicillin–streptomycin mixture and 20% fetal bovine serum (FBS)). 2. 0.25% Trypsin-EDTA. 3. Phosphate-buffered saline (PBS). 4. Ficoll-Hypaque gradient (for human MSC isolation from harvested bone marrow). 5. Sterile mortar and pestle (for murine MSC isolation).

15

Tracking Inflammation-Induced Mobilization of Mesenchymal Stem Cells

175

Table 1 Common MSC surface markers for human and murine MSC MSC surface markers Human

Murine

+

−

+

−

CD90

CD45

Sca-1

CD45

CD105

CD34

CD44

CD34

CD44

CD31

CD106

CD31

CD73

CD11b

CD140b

CD11b

CD166

C-Kit

CD146 CD140b GD2 CD271a a

CD271 is only found on freshly isolated human MSC, not on cultured MSC

6. 3 mg/ml Collagenase Type I (Worthington Biochemical) in α-MEM. 7. 180 cm2 Tissue culture dish. 8. Conjugated CD11b antibody (for murine MSC isolation— during immunodepletion step). 9. See Table 1 for list of additional antibodies used for MSC characterization. 2.2. MSC Differentiation 2.2.1. MSC Adipocyte Differentiation

1. Adipogenic induction medium (Dulbecco’s Modified Eagle’s Medium (DMEM), 10% FBS, penicillin, streptomycin, L-glutamine, 10 μg/ml insulin, 500 μM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 200 μM indomethacin). 2. Adipogenic maintenance medium: DMEM, 10% FBS, penicillin, streptomycin, L-glutamine, and 10 μg/ml insulin. 3. Isolated MSC. 4. 12-Well cell culture plate. 5. 10% Formalin. 6. Isopropanol (100 and 60%). 7. Oil Red O (Sigma) solution: Prepare Oil Red O working solution immediately prior to staining. 4 ml Water + 6 ml of 0.35%

176

E.L. Spaeth et al.

Oil Red O dissolved in 100% isopropanol. Filter twice with a 0.22 μm syringe filter. 2.2.2. MSC Osteoblast Differentiation

1. Isolated MSC. 2. 12-Well cell culture plate. 3. OsteoDiff Medium for osteoblast differentiation (Miltenyi Biotec, Auburn, CA). 4. Chromogenic alkaline phosphatase substrate BCIP/NBT (SIGMA FAST). 5. Alizarin Red S solution. 6. 100% Methanol. 7. PBS.

2.2.3. MSC Chondrocyte Differentiation

1. Isolated MSC. 2. 15 ml Polypropylene Falcon tube. 3. Chondrocyte differentiation medium: DMEM, penicillin, streptomycin, L-glutamine, 50 μg/ml ascorbic acid, 100 nM dexamethasone, and 10 ng/ml transforming growth factor β3 (TGF-β3). 4. PBS. 5. Formalin. 6. Histology core or processing capabilities. 7. Xylene. 8. 100% Alcohol (and dilutions 95, 90, 80, 70%). 9. 1% Alcian blue in 5% acetic acid. 10. Distilled water. 11. Nuclear fast red (Lab Vision Corporation). 12. Aqueous mounting medium.

2.3. In Vivo Mobilization and Tracking

1. αMEM with supplements (L-glutamine and penicillin– streptomycin mixture and FBS): 0–2% (for starvation) or 20% (for normal MSC). 2. 0.25% Trypsin-EDTA. 3. PBS. 4. Labeled tumor cell line of choice and MSC. 5. LipofectAmine and PLUS Technologies) (optional).

transfection

reagents

(Life

6. Plasmid-encoding GFP or other markers (optional). 7. CO2-independent medium (Optional). 8. Xenogen IVIS bioluminescence/fluorescence optical imaging system (Caliper Life Sciences).

15


177

9. IVIS Living Image software (Caliper Life Sciences). 10. Isofluorane. 11. In vivo source—e.g. mouse. 12. Insulin syringe (1 cm3; 28 G ½). 13. Chemiluminescent substrate reagent depends on the bioluminescent expression system in your cells. Most common luciferase substrates: —

—

2.4. Mouse Bone Marrow Transplantation

Renilla requires colenterazine (40 mg/ml) (Biotium, Inc.) to prepare—resuspend in methanol. Firefly requires D-luciferin firefly-potassium salt (125 mg/ kg) (Biosynth).

1. Ubiquitously expressing GFP and RFP transgenic mice. 2. Insulin syringe. 3. Mouse restraint device for tail vein injection or isoflurane for ocular vein injection. 4. Sterile PBS. 5. Clear, Lucite box with air holes. 6. Approved radiation facility. 7. Antibodies for FACS sorting MSC from bone marrow population (investigator’s discretion).

2.5. Immunohistochemisrty/Immunofluorescence/ Multispectral Imaging

1. Tissues stored in formaldehyde or snap-frozen in liquid nitrogen and stored at −80°C. 2. OTC compound (Miles, Inc.) or formaldehyde. 3. Sodium citrate buffer: 10 mM sodium citrate, 0.05% Tween 20, pH 6.0. 4. Blocking buffer: 3% BSA, 1% FBS, and 1% species of secondary antibody (see Note 18). 5. Wash buffer: T-PBS: PBS with 0.5% Tween-20. 6. Antibodies of choice—extensive multicolor staining resources can be acquired elsewhere. 7. Hematoxylin-eosin and immunohistochemical (IHC) staining kits for IHC and fluorescently labeled antibody for choice for IF. 8. Research optical microscope with reflected and transmitted light sources (CRi Nuance attachment (Caliper Life Sciences) for multispectral imaging can alleviate concerns of background autofluorescence and multiple markers, but is not necessary). 9. A high resolution digital camera. 10. Adobe Photoshop, Slidebook, or InForm Software can be used to analyze image data.

178

E.L. Spaeth et al.

3. Methods 3.1. MSC Isolation and Characterization

3.1.1. MSC Isolation (Human)

While validated MSC cell lines can be purchased from the ATCC, they are also easily isolated from human or mouse (as explained below). Briefly, harvested bone marrow can be plated on plastic and the population of adherent cells that grows out from is enriched in the cell population we define as MSC. Phenotypic characterization by flow cytometry is one method to validate MSC population although there is no consensus on a single set of markers. Table 1 lists the most commonly used markers; however, those listed are not all inclusive nor should they be used exclusively to define MSC. The other standard to define MSC is to analyze the differentiation potential. Adipocyte, osteoblast, and chondrocyte differentiation assays are briefly discussed in this chapter (Fig. 1). 1. Collect clinical bone marrow sample (according to institutional protocol). 2. Separate mononuclear cells by centrifugation over FicollHypaque gradient (Sigma, St. Louis, MO). 3. Plate at initial seeding density of 1 × 106 cells/cm2. Size of tissue culture plate/flask can vary by experimental conditions. 4. After 3 days, remove the non-adherent cells by washing with PBS. 5. Culture adherent cell monolayer until confluency. 6. Trypsinize (0.25% trypsin with 0.1% EDTA) cells and subculture at densities of 5,000–6,000 cells/cm2. 7. Use cell passages 3–4 for the experiments (see Note 1).

3.1.2. MSC Isolation (Murine)

1. Anesthetize and sacrifice mouse according to institution approved protocol (see Note 2). 2. Remove the two hind limbs (femur and tibia) and the iliac crest (hip) (see Note 3). 3. Place clean bones in warmed αMEM (no serum needed—in a Petri dish or conical tube). 4. When all bones are removed, in sterile environment, fill an 18 G needle with serum media, cut the ends (both proximal and distal) of the bone and insert the needle to flush marrow out into a new, sterile tissue culture dish with warmed, serum (20%) αMEM medium. 5. Flushed bones can be crushed with mortal and pestle in warmed media. 6. Spin-down crushed bone (at 250 g) and resuspend in 3 mg/ ml Collagenase Type I 2 ml/mouse, and placed in a 37°C shaker at 50 × g for 45 min.

15


179

Fig. 1. MSC characterization based on flow cytometry and differentiation assays. (a) Freshly isolated murine bone marrow sample stained with CD45. The subpopulation that contains MSC is found within the CD45 negative gate. Additional markers can be applied to the samples to select a more specific population. (b) First passage human MSC and freshly isolated murine MSC in culture are fibroblastic in appearance. (c) Adipogenic differentiation assay stains lipids with Oil Red O. Osteogenic differentiation assay has two stains, one to depict alkaline phosphatase activity and one to detect calcium deposits. Chondrogenic differentiation assay uses Alcian blue to detect mucopolysaccharides associated with chondroblast differentiation.

7. After incubation, bones were filtered out and cells were washed in PBS and added to the sterile culture dish in 20% αMEM. 8. Incubate at 37°C for about 3–5 days. 9. Discard supernatant and floating cells, culture adherent cell monolayer (MSC) until confluent.

180

E.L. Spaeth et al.

10. Trypsinize (0.25% trypsin with 0.1% EDTA) cells and subculture at densities of 5,000–6,000 cells/cm2. Size of tissue culture plate/flask can vary by experimental conditions. 11. Use cell passages 3–4 for the experiments (see Note 4). 3.2. Cell Differentiation Assays 3.2.1. Adipocyte Differentiation

1. Plate 2 × 104 MSC in a 12-well plate. 2. After cells reach confluence, change the medium to adipogenic induction medium for 72 h. 3. Change medium to the adipogenic maintenance medium for 24 h. 4. Repeat steps 2 and 3, induction/maintenance three times. 5. At the end of the third round, continue the adipogenic maintenance medium for 10 days changing the medium two times per week. 6. Fix adipocytes in 10% formalin for 1 h. 7. Wash adipocytes with 60% isopropanol and dry. 8. Lipid vacuoles are stained with Oil Red O solution. Place on dry adipocytes for 10 min. 9. Adipocytes and red lipid vacuoles can be imaged on any available microscope.

3.2.2. Osteogenic Differentiation

1. Plate 2.2 × 104 MSC subconfluently in a 12-well plate in OsteoDiff Medium. 2. Culture cells for 3 weeks, change medium 2× per week, with a volume of 1 ml per well. 3. Harvest cell culture between day 21 and day 30. 4. Fix cells with ice cold 100% methanol for 5 min then wash with PBS. 5. To visualize alkaline phosphatase activity, add alkaline phosphatase substrate to the well number 1 and incubate at 37°C for 10 min. Then, wash with water. 6. To visualize calcium deposits, add Alizarin Red S solution to the well number 2 and incubate at room temperature for 10 min. Then, wash with water. 7. Osteoblasts can be visualized on microscope of choice.

3.2.3. Chondrogenic Differentiation

1. Pellet 3.5 × 105 MSC in a 15 ml Falcon tube at 250 × g. 2. Resuspend the cells in chondrocyte differentiation medium. 3. Pellet suspension again and place into culture as a pellet— loosen the cap on the 15 ml Falcon tube for gas exchange. 4. Add 10 ng/ml TGF-β3 daily. Change chondrocyte differentiation medium three times per week—do not disturb the pellet.

15


181

5. After 21 days, rinse pellet in PBS and fix in 10% formalin. 6. Paraffin embed and section for histology. 7. Deparaffinize in xylene and rehydrate in a series of alcohols. 8. To detect mucopolysaccharides associated with chondroblasts, stain with 1% Alcian blue in 5% acetic acid for 30 min. 9. Rinse with distilled water. 10. Counterstain with Nuclear Fast Red. 11. Mount in aqueous mounting medium. 12. Visualize on any microscope available. 3.3. In Vivo Mobilization and Migration of MSC

3.3.1. Tracking Exogenously Injected MSC Migration Towards Tumor

The therapeutic utilization of MSC has been attempted in numerous applications. It is well understood that MSC migrate towards sites of inflammation. Inflamed tissues produce factors that attract MSC to aid in wound repair. In this chapter, the site of inflammation that will be thoroughly addressed is the tumor microenvironment; however, MSC have been shown to migrate to other tissue environments including ischemic heart, ischemic brain, subcutaneous incision, and bone fracture (24–27). Location of tumor engraftment will depend on the tumor subtype as well as on analytical method at hand and can be placed subcutaneously, intravenously, intraperitoneally, or intracranially. Herein, we review three techniques that improve the in vivo analysis of MSC migration. Live cell bioluminescent (or fluorescent) imaging allows investigators to follow MSC migration within a live animal without the need to sacrifice it. Transgenic mice like ubiquitously expressing or promoter-specific expressing fluorescent-colored mice can be used to follow endogenous migration of MSC without exogenous manipulation of the system. This simple technique can be modified to address multiple questions. Finally, multispectral immunohistochemical/immunofluorescence imaging is a method that can be used to analyze complex histological markers in combination with one another. 1. First, the tumor cell line of choice needs to be stably labeled using a lenti or retroviral system. For this example, our system will label the breast cancer cell line, 4T1 with a GFP-tagged renilla-luciferine (GFP+/rLuc+) construct. Fluorescent Acquired Cell Sorting (FACS) sort labeled cells and expand for engraftment (see Note 5). 2. Prepare an adequate number of tumor cells for orthotopic tumor engraftment of 1 × 105 cells per injection in Nod-Scid mice (see Note 6). 3. Grow and label MSC in vitro similarly to tumor cell labeling but use an alternative luciferin construct (e.g., RFP-labeled firefly luciferin) (see Note 7).

182

E.L. Spaeth et al.

4. Engrafted tumors should be monitored weekly until appropriate size (see Note 8), larger than 5 mm diameter ensures visible and palpable tumor target. 5. To image rLuc + 4T1 in vivo, coelenterazine should be prepared immediately prior to injection. Substrate should be kept on ice and in the dark to maintain efficacy. Injections can be given intravenously or intraperitoneally—i.v. injections are more sensitive but take longer to complete and given the short half-life of the luciferase substrate. 6. Once tumors are adequate in size. Prepare MSC: 1 × 106 cells per injection resuspended in 100 μl of PBS. Intravenously inject MSC into Scid mice (see Note 9). 7. MSC can be imaged 0–3 h following injection to confirm the presence of MSC, which will be primarily in the lungs of the mice. The imaging substrate will be D-luciferin for the ffLuclabeled MSC, and should be prepared on ice and kept in the dark until IP injection similar to the coelenterazine. 8. To catch peak MSC recruitment to the tumor site, mice should be imaged every 24 h post MSC injection. Peak recruitment is between 48 and 72 h (Fig. 2). But imaging can be continued according to investigator’s discretion (see Note 10). 9. This system of exogenous recruitment of circulating MSC can be applied to many other injury/inflammatory models systems including cutaneous lacerations, muscular lacerations, bone fractures, and ischemic heart or brain models. This model can also be carried out using fluorescent cells instead of bioluminescent ones; however, there are a few limitations of the fluorescence model: (1) a nude, or shaved mouse is better; (2) autofluorescence can be a limitation when using fluorescent markers on the lower end of the spectrum; (3) depth of the tumor/signal within the animal can also be a problem that compounds the autofluorescent effect—the fluorescence may never be detected. 3.3.2. Mobilization of MSC Towards the Tumor Microenvironment

In this example, we address this question of endogenous MSC mobilization to the tumor microenvironment by using two fluorescently labeled transgenic C57/B6 mice: GFP (green) and RFP (red) (28). 1. Syngenic bone marrow transplant C57/B6 recipients should be between 6 and 10 weeks of age when lethally irradiated. Briefly, irradiate mice with a single dose of 9.5 Gy 4 h before donor bone marrow reconstitution (see Note 11). 2. Donor mice should be sacrificed and bone marrow collected. RFP-MSC from RFP transgenic bone marrow will be isolated and sorted by FACS. Briefly, bone marrow from the tibia, fibia, and iliac crest of the mice should be flushed, crushed with mortal and pestle, and resuspended in collagenase I for 1 h at

15


183

Fig. 2. Bioluminescent images using luciferin substrates. (a) r-luc expressing 4T1 in a 96-well plate can be imaged. Average bioluminescent radiance observed can be plotted against number of tumor cells per well showing a linear correlation. (b) 2 h Post i.v. injection of mMSC shows bioluminescent activity in the lungs and at the injection sites in the tail. (c) In vivo image of murine breast cancer line, 4T1 labeled with r-luc on the left. Labeled tumor cells luminesce in the limbs of the mouse (in the inguinal adipose tissue where the two intra-fat tumor injections were initially given) and in the lungs (sites of metastases). On the right panel, ff-luc labeled mMSC 3 days after injection. The luminescent areas are dimmer, but overlap with the bilateral tumors as indicated by the arrowheads.

37°C at 50 × g (see MSC isolation in Subheading 3.1). Suspension can be collected and filtered (40 nm cell filter) before being labeled for FACS analysis (see Note 12). 3. Donor GFP transgenic bone marrow will be collected and depleted for MSC in the identical manner as described above and will be called the “non-MSC” bone marrow population. 4. Bone marrow fractions collected and isolated from either the GFP donor or the RFP donor will be admixed in vitro in a tube. Cell mixture ratio for one i.v. injection for one mouse will consist of 1 × 106 (can use between 1 × 106 and 1 × 107 bone

184

E.L. Spaeth et al.

marrow cells) GFP “non-MSC” bone marrow plus 1 × 106 RFP-MSC. See Fig. 3 for a diagram of the procedure. Conversely, alternative controls can be prepared using 1 × 106 RFP “non-MSC” bone marrow plus 1 × 106 GFP-MSC or the “non-MSC” populations alone (see Note 13).

Fig. 3. Diagram of bone marrow transplant schematic. Recipient mice are non-fluorescent and will be lethally irradiated 3–6 h prior to bone marrow re-derivation. Meanwhile, donor mice will be sacrificed and bone marrow will be collected and sorted by FACS to retrieve an “MSC” population derived from the RFP mouse and a total bone marrow minus MSC (or “nonMSC”) population from the GFP mouse. These populations will then be admixed according to an assay-dependent ratio and intravenously injected into the recipient mouse. The GFP/RFP bone marrow re-derivation can be confirmed after 21 days by flow cytometric analysis of drawn blood sample. Upon confirmation, tumors can be engrafted.

15


185

5. Four hours following lethal irradiation, sorted RFP and GFP bone marrow mixtures will be i.v. injected into the recipient mouse (see Note 14). 6. Three weeks following bone marrow transplantation, the control mouse that received PBS alone will have died. Blood can be drawn from the mouse and analyzed by flow cytometry to confirm the presence of the fluorescent bone marrow. 7. Now that we have a C57/B6 mouse with GFP-labeled “nonMSC” or hematopoietic system and RFP-labeled MSC, we can engraft tumors into the mouse to elucidate the contributions of both the MSC and the “non-MSC” components that mobilize in response to tumor engraftment. The tumor model used in this model is the EO771, a C57/B6 breast tumor. Subcutaneous or orthotopic injections of 5 × 104 EO771 tumor cells will engraft into substantial 10–20 mm diameter tumors within three to five weeks. 8. Mice can be sacrificed at any number of time points leading up to the endpoint of the experiment. At time of sacrifice, tumors, organs, and other tissues can be collected for analysis by flow cytometry to quantify the number of RFP versus GFP cells are found within the tumor (or organ in question). The tissues can also be collected for immunofluorescence and/or immunohistochemistry, for which they can be snap frozen in OTC compound on dry ice and 100% ethanol, or they can be fixed in formalin (see Note 15). 9. This syngenic model with fluorescently labeled endogenous cells can be applied to many different models of inflammation including those previously mentioned like subcutaneous incisions or ischemic tissue models. The potential of syngenic models such as these leaves great potential for the elucidation of MSC as an important stromal cell involved in wound healing (see Note 16). 3.3.3. Imaging of Tumor Sections

1. The slides from the aforementioned bone marrow transplant experiment can be analyzed by immunofluorescent (IF) or IHC depending on the microscopy system available to the investigator. In this section, we focus mainly on IF with a multispectral imaging camera and software (CRi Inc., Woburn, MA) that enables the investigator to identify true fluorescence within high background tissue autofluorescence as well as identify multiple colors based on precise wavelength emission differences that might not be separated by a normal fluorescent microscope camera (see Note 17). 2. Tumor sections from paraffin-embedded blocks can be sliced in 5 nm sections. Briefly, slides can be deparaffinized in a series of xylene and ethanol washes prior to antigen retrieval for 20 min in boiling sodium citrate buffer. Tumor sections can alternatively be snap frozen instead of formalin preserved.

186

E.L. Spaeth et al.

3. Incubate slides in blocking buffer (see Note 18) for 30 min at room temperature (or overnight at 4°C). 4. If using an antibody, prepare dilution as per manufacturer’s instruction in blocking buffer. Incubate for 2 h at room temperature (or overnight at 4°C). In this example, we will be using a rabbit anti-RFP at a 1:200 dilution (see Note 19). 5. Wash in T-PBS for 5 min (3×). 6. Prepare a fluorescently labeled secondary antibody solution in blocking buffer at a 1:1,000 dilution. In this case, we will use goat-anti-rabbit Alexafluor 594 for 1 h at room temperature. The slides should be kept covered during this step. 7. Wash as done in step 5. 8. DAPI stain nucleus for 1 min with 5 mg/ml DAPI stock solution diluted 1:10,000. 9. Wash as done in step 5. 10. Rinse slides in water before applying fluorescent mounting media and a 1½ cover slip (170 nm thickness for optimal fluorescent image quality). 11. Let dry (protect from light) and apply nail hardener to the edges of the slide. 12. Imaging can be done on a microscope with a proper CRi Nuance camera attachment according to manufacturer’s manual. Figure 4 shows the image of a tumor section using the system described herein. 13. Data analysis software InForm (CRi Inc., Woburn, MA) allows for quantification of fluorescence-labeled cells within the image sections and can quantify based on cellular location or co-localization with additional color (see Note 20).

4. Notes 1. Cells can be sorted by flow cytometry for human MSC markers if a subpopulation is desired (CD44+, CD90+, CD105+, CD73+, CD166+, CD146+, CD140b+, and CD34−, CD45−). 2. Remove bone immediately after sacrificing the mouse. 3. Remove as much muscle tissue, skin, and fur from the bone using a scapula to scrape the remaining tissue away before placing in warmed αMEM. 4. Cells can be sorted by flow cytometry for mouse MSC markers if a subpopulation is desired (CD44+, Sca1+, NG2+ and CD34−, CD45−).

15


187

Fig. 4. Immunofluorescent image of EO771 breast cancer tumor section. The Alexa594 staining for a stromal marker (big arrowheads) is depicted in white in the smaller, unmixed image and black in the large multispectral image. GFP cells (thin arrows) within the tumor are depicted in white in the smaller unmixed image, and in dark gray in the large multispectral image. DAPI nuclear staining is depicted in white in the smaller unmixed image and in light gray in the large multispectral image.

5. The tumor model system can be xenograft (human tumor and human MSC) or syngenic (murine tumor and murine MSC) depending on the system of choice. Because many transgenic mouse models exist, the potential for studying the mobilization of gene-modified (either +/− geneX) MSC towards the tumor microenvironment is extensive. 6. Number of tumor cells injected depends on the system in use. To get a significant (1–10 mm diameter tumor) of 4T1 cells in about 4 weeks, inject 2 × 105 cells per subcutaneous or orthotopic tumor site in 100 μl PBS. About 8 × 106 4T1 per T-175 flask at 85% confluency. 7. Start expanding MSC early enough to achieve 1 × 106 MSC per intravenous injection. MSC are readily labeled with lentiviral vectors according to common protocols. About 3 × 106 MSC per T-175 flask at 80% confluency.

188

E.L. Spaeth et al.

8. In these methods, the IVIS System from Caliper Life Sciences will be used, alternative bioluminescent and fluorescent live imaging systems can be used including Kodak’s In Vivo Multispectral Imaging System by Carestream or LI-COR’s Pearl Imager. 9. Intravenous injections can be given via lateral tail vein or ophthalmic plexus routes. The latter method requires the mouse to be anesthetized in order to insert the needle on the inner side of the ocular cavity, whereas the former method requires a restraint device, but no anesthetic. 10. If your study permits, mice can be sacrificed at any number of time points following MSC injection to confirm the in vivo bioluminescent MSC detection with more conventional IHC or FACS analysis of tumor sections to confirm the presence of MSC. 11. Syngenic bone marrow transplant requires good planning, and long time commitment to complete and will vary depending on the number of animals in the study. 12. Because the surface markers for MSC are still controversial, a select number of markers can be used based on the investigators discretion. Negative gating on CD45 and CD11b eliminate the hematopoietic and macrophage lineages, gating on the positive markers of known MSC subsets like NG2, PDGFRβ, or Sca-1 can be further used to derive a population of MSC. However, this population is not all inclusive. Collect both gated populations. These populations will be known as the “MSC” and “non-MSC” bone marrow fractions. 13. Bone marrow from the tibia, femur, and iliac crest of one mouse will give 3 × 107 total bone marrow cell, but only 1–3 × 106 MSC, therefore to prospectively isolate 1 × 106 MSC for each recipient mouse, you need at 3–5 donor mice per recipient. Alternatively, number of MSC used per bone marrow transplant are at the investigator’s discretion. The ratio of MSC to total bone marrow can be 1:1 or 1:100 (1:100 is a more biologically realistic ratio). 14. Intravenous injections can be given via lateral tail vein or ophthalmic plexus routes. The bone marrow mixture of RFP-MSC and GFP non-MSC or vice versa will be resuspended in 100 μl of PBS. As a control, one mouse will receive PBS alone. 15. Both frozen and paraffin-embedded tissue sections have been shown to produce good naïve images of fluorescent-labeled cells. 16. The most significant limitation is one of MSC characterizations. The heterogeneous population of MSC and the lack of defined surface markers or protein expression leaves a lot of room for interpretation. Therefore, initially defining your

15


189

population of “MSC” is significant to the outcome of your experiment. As MSC are further studied, the use of MSCspecific promoters to drive reporter expression specific to MSC will be of great use in this model, where we will no longer need to prospectively isolate MSC based on unrefined marker expression. 17. If you naïve fluorescence is not visible under the microscope, respective fluorescent antibodies can be used to enhance the visualization of the fluorescently labeled cell. 18. For blocking buffer, you can use serum from the species that your secondary antibody is made in, e.g., using a goat-antirabbit Alexa Fluor secondary, use normal goat serum in the blocking buffer. 19. Proper controls are important to the experiment. When working with a multicolored tissue, it is good to have single colored controls for each color in use. For example, our final slide will contain four colors: DAPI, Alexa647, GFP, and RFP. Therefore, we will need five control slides. One background slide of tumor only to account for background tissue autofluorescence. One tumor slide with RFP cells present only. One tumor slide with GFP cells present only. One tumor slide with DAPI only and one tumor slide with Alexa647 only. These controls will allow us to set up a proper “library” of positive controls for our final, multicolor slide. 20. These methods are not limited to the CRi system, any functional fluorescent microscope can be used to suite the investigators needs. References 1. Spaeth E, Klopp A, Dembinski J, Andreeff M, Marini F (2008) Inflammation and tumor microenvironments: defining the migratory itinerary of mesenchymal stem cells. Gene Ther 10:730–738 2. Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H (2008) Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol 180: 2581–2587 3. Wu Y, Chen L, Scott PG, Tredget EE (2007) Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells 25:2648–2659 4. Li H, Fu X, Ouyang Y, Cai C, Wang J, Sun T (2006) Adult bone-marrow-derived mesenchymal stem cells contribute to wound healing of skin appendages. Cell Tissue Res 326: 725–736

5. Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff M (2002) Bone marrowderived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res 62:3603–3608 6. Dwyer RM, Potter-Beirne SM, Harrington KA, Lowery AJ, Hennessy E, Murphy JM et al (2007) Monocyte chemotactic protein-1 secreted by primary breast tumors stimulates migration of mesenchymal stem cells. Clin Cancer Res 13:5020–5027 7. Coffelt SB, Marini FC, Watson K, Zwezdaryk KJ, Dembinski JL, Lamarca HL et al (2009) The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells. Proc Natl Acad Sci USA 106:3806–3811 8. Sordi V, Malosio ML, Marchesi F, Mercalli A, Melzi R, Giordano T et al (2005) Bone marrow mesenchymal stem cells express a restricted set

190

E.L. Spaeth et al.

of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood 106:419–427 9. Kim SM, Lim JY, Park SI, Jeong CH, Oh JH, Jeong M et al (2008) Gene therapy using TRAIL-secreting human umbilical cord bloodderived mesenchymal stem cells against intracranial glioma. Cancer Res 68:9614–9623 10. Sonabend AM, Ulasov IV, Tyler MA, Rivera AA, Mathis JM, Lesniak MS (2008) Mesenchymal stem cells effectively deliver an oncolytic adenovirus to intracranial glioma. Stem Cells 26:831–841 11. Ren C, Kumar S, Chanda D, Kallman L, Chen J, Mountz JD et al (2008) Cancer gene therapy using mesenchymal stem cells expressing interferon-beta in a mouse prostate cancer lung metastasis model. Gene Ther 15:1446–1453 12. Khakoo AY, Pati S, Anderson SA, Reid W, Elshal MF, Rovira II et al (2006) Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi’s sarcoma. J Exp Med 203:1235–1247 13. Kanehira M, Xin H, Hoshino K, Maemondo M, Mizuguchi H, Hayakawa T et al (2007) Targeted delivery of NK4 to multiple lung tumors by bone marrow-derived mesenchymal stem cells. Cancer Gene Ther 14:894–903 14. Hung S, Deng W, Yang WK, Liu R, Lee C, Su T et al (2005) Mesenchymal stem cell targeting of microscopic tumors and tumor stroma development monitored by noninvasive in vivo positron emission tomography imaging. Clin Cancer Res 11:7749–7756 15. Hata N, Shinojima N, Gumin J, Yong R, Marini F, Andreeff M et al (2010) Platelet-derived growth factor BB mediates the tropism of human mesenchymal stem cells for malignant gliomas. Neurosurgery 66:144–156 16. Spaeth EL, Dembinski JL, Sasser AK, Watson K, Klopp A, Hall B et al (2009) Mesenchymal stem cell transition to tumor-associated fibroblasts contributes to fibrovascular network expansion and tumor progression. PLoS One 4:e4992 17. Kidd S, Spaeth E, Klopp A, Andreeff M, Hall B, Marini FC (2008) The (in) auspicious role of mesenchymal stromal cells in cancer: be it friend or foe. Cytotherapy 10:657–667 18. Nakamura K, Ito Y, Kawano Y, Kurozumi K, Kobune M, Tsuda H et al (2004) Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther 11:1155–1164

19. Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, Bekele BN et al (2004) Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst 96:1593–1603 20. Kidd S, Caldwell L, Dietrich M, Samudio I, Spaeth EL, Watson K et al (2010) Mesenchymal stromal cells alone or expressing interferon-beta suppress pancreatic tumors in vivo, an effect countered by anti-inflammatory treatment. Cytotherapy 12:615–625 21. Ju S, Teng G, Zhang Y, Ma M, Chen F, Ni Y (2006) In vitro labeling and MRI of mesenchymal stem cells from human umbilical cord blood. Magn Reson Imaging 24:611–617 22. Gonzalez-Lara LE, Xu X, Hofstetrova K, Pniak A, Chen Y, McFadden CD et al (2011) The use of cellular magnetic resonance imaging to track the fate of iron-labeled multipotent stromal cells after direct transplantation in a mouse model of spinal cord injury. Mol Imaging Biol 13:702–711 23. Lo Celso C, Fleming HE, Wu JW, Zhao CX, Miake-Lye S, Fujisaki J et al (2009) Live-animal tracking of individual haematopoietic stem/ progenitor cells in their niche. Nature 457:92–96 24. Chen D, Zhang Z, Wu X, Lin JH (2007) Distribution of intravenously grafted bone marrow mesenchymal stem cells in the viscera tissues of rats before and after cerebral ischemia. J Clin Rehab Tissue Eng Res 11: 10160–10164 25. Barbash IM, Chouraqui P, Baron J, Feinberg MS, Etzion S, Tessone A et al (2003) Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation 108:863–868 26. Wu GD, Nolta JA, Jin YS, Barr ML, Yu H, Starnes VA et al (2003) Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation 75:679–685 27. Kidd S, Spaeth E, Dembinski JL, Dietrich M, Watson K, Klopp A et al (2009) Direct evidence of mesenchymal stem cell tropism for tumor and wounding microenvironments using in vivo bioluminescence imaging. Stem Cells 10:2614–2623 28. Kidd S, Spaeth E, Watson K, Burks J, Lu H, Klopp A et al (2012) Origins of the tumor microenvironment: quantitative assessment of adipose-derived and bone marrow-derived stroma. PLoS One 7:e30563