Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.
Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention.
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation.
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.
The tumor microenvironment actively participates in most, if not all, aspects of tumorigenesis and cancer progression, including metastasis formation and the development of resistance to therapeutics1. This stresses the need for preclinical orthotopic cancer mouse models that allow dissection of the complex tumor-stroma interactions occurring in the tumor niche.
Among the many cellular components of the tumor microenvironment, mesenchymal stem cells (MSCs) strongly contribute to cancer progression in multiple cancer types such as breast cancer, prostate cancer, brain tumors, multiple myeloma, and osteosarcoma2,3,4,5,6,7. MSCs are multipotent stem cells that reside in various adult and fetal tissues, including bone marrow, adipose tissue, placenta, umbilical cord blood, and others8,9. In response to cancer-generated inflammatory signals, MSCs migrate towards tumor sites, incorporate into the tumor microenvironment and ultimately differentiate into cancer-supporting cells10. These cancer-associated MSCs provide essential factors (i.e., growth factors, chemokines, cytokines, and immunosuppressive mediators) for tumor progression acting both on tumor cells and on the surrounding stroma2,3,11,12,13. While the tumor-promoting effects of cancer-associated MSCs have been investigated in numerous cancer models, the mechanisms by which tumor cells reprogram MSCs to shape a cancer-promoting niche are poorly understood. Here we describe the generation of an orthotopic xenograft model that specifically allows the study of the pro-tumorigenic interaction between bone cancer cells and MSCs via extracellular vesicles (EVs).
EVs are crucial mediators of intercellular communication between tumor and stromal cells14. EVs carry functional biomolecules of the cell of origin, including proteins, lipids, and regulatory RNAs. Once released in the extracellular space, these vesicles can be taken up by surrounding cells or carried to distant sites via the blood or the lymphatic circulation, and can profoundly influence target cell behavior.15,16,17 For instance, uptake of cancer EVs by stromal fibroblasts may result in myofibroblast differentiation supporting angiogenesis and accelerating tumor growth in vivo18,19, internalization by endothelial cells can stimulate tumor angiogenesis and increase vascular permeability16,20, and interaction with immune cells might lead to suppression of the antitumor immune response21.
We recently demonstrated, using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, that tumor cells release high amounts of EVs that prompt MSCs to acquire a pro-tumorigenic and pro-metastatic phenotype. This effect is due to a dramatic change in the MSC cytokine expression profile (referred to as "MSC education"), and can be prevented by the administration of a therapeutic interleukin-6 receptor (IL-6R) antibody7. Our work demonstrated that cancer EVs are crucial modulators of MSC behavior, thus providing a rationale for microenvironment-targeted approaches to halt osteosarcoma progression. Herein, we describe a step-by-step protocol to investigate the EV-mediated tumor-MSC interaction in vivo. This model is intended to: 1) specifically define the cancer EV-induced alterations of MSC behavior in the tumor microenvironment, 2) evaluate how this interaction contributes to bone tumor growth and metastasis formation, and 3) study whether interfering with the EV-mediated crosstalk in vivo prevents cancer progression.
Human adipose tissues for mesenchymal stem cell isolation were obtained from the department of Plastic Surgery of the Tergooi Hospital (Hilversum, Netherlands) after approval by the Institutional Ethical Committee and written informed consent. GFP-positive adipose MSCs were obtained from the Department of Medical and Surgical Sciences for Children and Adults (University of Modena and Reggio Emilia).
Animal experiments were performed in accordance with the Dutch law on animal experimentation, and the protocol was approved by the committee on animal experimentation of the VU University medical center, Amsterdam, Netherlands.
1. Isolation of Tumor-Secreted Extracellular Vesicles.
2. EV Characterization.
Purified EVs can be visualized by transmission electron microscopy (TEM)23.
3. Education of MSCs by tumor-derived EVs.
4. EV Internalization Assay.
To visualize EV uptake by MSCs, label EVs with a green-fluorescent linker dye (GFLD) (commercially available) following the manufacturer's protocol:
5. Assessment of MSC Cytokine Expression Profile.
6. Generation of an Orthotopic Xenograft Mouse Model of Osteosarcoma.
7. Assessment of Lung Nodule Number
8. Histological Analysis
In this study, we explored the ability of osteosarcoma-secreted EVs to educate MSCs towards a pro-tumorigenic and pro-metastatic phenotype. We show that osteosarcoma cells release exosome-like EVs that are internalized by MSCs. We measured the alteration of the MSC cytokine expression profile induced by cancer EVs, and evaluated the effect of the EV-educated MSCs on tumor growth and metastasis formation. The general representation of the study design is illustrated in Figure 1.
EVs released by 143B osteosarcoma cells or control human fibroblasts were purified by differential centrifugation. EV purity was confirmed by electron microscopy, which revealed that the preparations mainly contained vesicles with a diameter ranging between 40 – 100 nm (Figure 2A). To assess whether cancer EVs interact with MSCs, we labeled the vesicles with a green-fluorescent lipophilic linker dye (GFLD) and incubated them overnight with the target cells. By fluorescence microscopy we observed efficient EV uptake by MSCs (Figure 2B). Moreover, flow cytometry analysis showed that osteosarcoma and control EVs are internalized with comparable efficiency (Figure 2C). To investigate whether osteosarcoma EVs alter the immunomodulatory properties of MSCs, we used a multiplex bead-based cytokine immunoassay, which showed that tumor EVs determine a 2-fold increase in IL-8 and IL-6 production compared with human fibroblast control EVs (Figure 2D,E).
To investigate whether osteosarcoma EVs educate MSCs to adopt a pro-tumorigenic and pro-metastatic phenotype, we employed a bioluminescent, orthotopic xenograft mouse model of osteosarcoma. All animal experiments were performed by one operator. Metastatic 143B-Fluc cells were transplanted into the tibia of athymic nude mice, and, after two days, osteosarcoma-xenografted mice were subjected to a single administration of GFP-positive EV-educated MSCs. Mice receiving naïve (non-educated) MSCs or no MSCs were used as control groups. No animals died before the described endpoints. We demonstrated by caliper measurement and bioluminescence imaging (BLI) that mice treated with EV-educated MSCs had accelerated tumor growth compared with control groups (Figure 3A). Representative images of tumor size of each treatment arm are shown in Figure 3C. Our data indicate that a single i.v. administration of educated MSCs affects tumor progression as early as day 10 after inoculation (Figure 3B). In addition, four days after systemic injection of educated/naïve GFP-expressing MSCs (Figure 4A), we could visualize GFP-expressing cells both in the bone marrow (Figure 4B) and in the tumor tissue (Figure 4C), demonstrating MSC homing to the tumor site.
Ex vivo BLI analysis of lungs, liver, kidney and spleen (data not shown) showed lung nodule formation exclusively in the lungs in mice of all treatment arms (Figure 4D-F). Strikingly, mice receiving EV-educated MSCs had higher number of lung metastases compared with mice receiving non-educated MSCs or no MSCs (Figure 4G), suggesting that within the tumor microenvironment educated MSCs increase the metastatic potential of osteosarcoma cells in vivo7.
Figure 1. Schematic representation of the study design. Human primary MSCs are educated with EVs isolated from cultured metastatic osteosarcoma (143B) cells. Cancer EV purity is assessed by electron microscopy, and internalization by MSCs is visualized by fluorescence microscopy and analyzed by flow cytometry. Changes in the MSC cytokine expression profile are evaluated by cytometric bead array (CBA). To define the role of EV educated MSC on tumor growth and metastasis formation, osteosarcoma-bearing mice are injected with EV-educated (or naïve) MSC. Tumor growth is followed by bioluminescence imaging (BLI) and caliper measurement, while metastasis formation is evaluated at the experimental end-point by ex vivo BLI and histological analysis. Please click here to view a larger version of this figure.
Figure 2. Purification of EVs and their interaction with MSCs. (A) Transmission electron microscopy micrograph of EVs isolated from 143B cells (scale bar: 100 nm). (B) Uptake of GFLD-labeled 143B EVs by MSCs assessed by fluorescence microscopy (scale bar: 10 µm). (C) Internalization of GFLD-labeled 143B EVs (orange) or control (human fibroblast, hf) EVs (green) by MSCs as analyzed by flow cytometry. (D) Multiplex detection of inflammatory cytokines in the supernatants of MSCs exposed to 143B (143B EV-MSC) or hf-EVs (hf EV-MSC) by cytometric bead array. 143B-MSC express higher levels of IL-6 and IL-8 compared to control (untreated and hF EV-treated) MSCs. The dotted lines indicate the shift in cytokine production. (E) IL-8 and IL-6 protein concentration in the conditioned media of EV-treated MSCs as analyzed by cytometric bead array, expressed as fold induction relative to the untreated control. Data are expressed as mean ± SD, n = 2. Please click here to view a larger version of this figure.
Figure 3. Bioluminescent, orthotopic xenograft mouse model of osteosarcoma. (A) Estimation of tumor volume using caliper measurements, and (B) tumor growth measured by bioluminescence imaging (BLI). Tumor size is expressed as photon flux (photons/sec). ** p < 0.01, non-educated MSC (n=6) vs educated MSC (n = 6), unpaired t-test. Data are expressed as mean ± SEM. (C) Representative BLI images of mice with established osteosarcoma tumors (top panel). Color-scale bar ranges from 7.7 x 107 (purple) to 2.6 x 108 (red) photons/sec. In the bottom panel, the tumor area is visualized without BLI overlay. Arrows indicate the tumor bulk. Scale bars: 0.6 cm. Please click here to view a larger version of this figure.
Figure 4. Imaging of MSCs and ex vivo lung tissue. (A) Fluorescence microscopy image of cultured GFP-positive adipose-derived MSCs (green). (B) Immunofluorescence staining of GFP-positive MSCs in the bone marrow and (C) in tumor tissue of MSC-receiving mice (green: MSCs, blue: DAPI stained nuclei; scale bar 10 µm). (D-F) Ex vivo bioluminescence imaging (BLI) showing higher number of metastatic foci in mice receiving EV-educated MSCs (F) as compared to mice receiving naïve MSC (E) or no MSC (D). Color scale bar ranges from 1 x 106 (purple) to 1.5 x 106 (red) photons/sec. (G) Quantification of lung metastasis number as visualized by BLI (Lines represent the median; * p < 0.05, one-tailed t-test). Please click here to view a larger version of this figure.
Tumor-secreted extracellular vesicles (EVs) can alter the physiology of local and distant mesenchymal cells to generate a tumor-supportive environment. Here we describe the generation of a preclinical mouse model of osteosarcoma that allows dissection of the EV-mediated interactions between tumor cells and mesenchymal stem cells (MSCs) in vivo. We show that systemic injection of human tumor EV-educated MSCs in mice bearing osteosarcoma xenografts strongly promotes cancer growth and metastasis formation by activating the IL-6/STAT3 signaling pathway7.
Recent studies have shown that cancer EVs can aid in the formation of the pre-metastatic niche by altering the behavior of local or bone marrow-derived stromal cells16,28,29. These studies have been mostly conducted by directly injecting cancer-derived vesicles in the recipient mouse, which complicates the identification of the cell types responsible for the tumor-promoting effects. In our protocol, the education of MSCs with cancer EVs occurs in vitro prior to MSC injection. This approach allows addressing of the specific contribution of the tumor-MSC crosstalk to cancer progression, and minimizes confounding indirect effects of cancer EVs on other components of the local or systemic environment.
To evaluate whether the educated MSCs home to the tumor site, we systemically injected GFP-positive MSCs in the tail vein of the tumor-bearing mice. Tail vein injections are a crucial, but technically challenging step in many experimental protocols. To ensure minimal variation among injections the procedure should be performed by experienced investigators. Correct intravenous administration can be visually confirmed by observing the movement of the fluid through the vein. If a white area appears on the tail or resistance is encountered during injection, vascular access was not achieved. In this case the procedure should be repeated by inserting the needle above the first injection site. Because MSCs readily home in on the tumor, successful administration of (GFP-positive) MSCs can be confirmed by immunofluorescence staining of the tumor tissue and bone marrow four days after injection (Figure 4B,C).
We show that cancer-secreted EVs induce a tumor-supportive phenotype in MSCs by altering the production of inflammatory cytokines. This finding is particularly relevant since immune modulation and tumor immune evasion are key mechanisms in malignant progression. Our data suggest that cancer EVs might directly, as reported by independent studies21,30,31,32,33, or indirectly (via MSCs) influence innate or adaptive immune components. However, the use of a xenograft (immunocompromised) model limits the possibility to investigate this aspect of EVs. Thus, similar approaches in immunocompetent or humanized mouse models need to be undertaken to define the role of the EV-mediated tumor-MSC-immune cell interactions in cancer.
Finally, because MSCs can be recruited from the bone marrow or other tissue sources to tumors, our method is not restricted to the study of bone cancers, but can be applied to investigate the role of tumor EV-educated MSCs in multiple cancer types2,3,4,5,6, as recent studies in melanoma and lymphoma models confirm34.
The authors have nothing to disclose.
S.R. Baglio was supported by a fellowship by Associazione Italiana per la Ricerca sul Cancro (AIRC) co-funded by the European Union,. In addition, this project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 660200 (to S.R. Baglio).
Equipment | |||||||
Ultra Centrifuge | Beckman | Optima L-90K | |||||
Rotor SW32Ti | Beckman | 369650 | Referred to in the manuscript as ultra-swinging bucket rotor | ||||
Transmission electron microscope | Zeiss | EM109 | Or similar TEM | ||||
Digital camera | Nikon | DMX 1200F | Or similar camera | ||||
Imaging software TEM | Nikon | ACT-1 | |||||
Fluorescence microscope | Zeiss | Imager.D2 | Or similar Fluorescence microscope | ||||
Imaging software FM | Zeiss | ZEN Blue | |||||
Incubator | Nuaire | 4750E | |||||
Centrifuge | Hettick | ROTANTA 460R | |||||
-80 Freezer | Thermo electro corporation | n.a. | |||||
FACS | BD | BD FACScalibur | Or similar flow cytometer | ||||
Drill | Ferm | FCT-300 | With 0.8 mm drill | ||||
HSS micro twist drills, 0.8 mm | Proxxon | 28 852 | 0.8 mm drill | ||||
IVIS camera | Xenogen | Ivis Lumina | Referred to in the manuscript as bioluminescence camera. Xenogen is now part of Perkin Elmer | ||||
Living image software2.60 | Xenogen / Igor Por | n.a | Xenogen is now part of Perkin Elmer | ||||
10 µL Syringe | Hamilton | Neuros Model 1701 RN | |||||
Needle: Hamilton RN Needle for Syringe, 26 Gauge, Pointstyle AS, custom length 2 cm | Hamilton | n.a. | |||||
Caliper | Mitutoyo | G08004463 | |||||
Autoclave | Astell | n.a. | |||||
Heat Lamp | Philips | n.a. | |||||
Culture media | |||||||
Fetal Bovine Serum | Hyclone | RYG35912 | |||||
Platelet Lysate | n.a. | n.a. | |||||
IMDM medium | Lonza | BE12-722F | |||||
alpha-MEM medium | Lonza | BE02-002F | |||||
DMEM medium | Lonza | BE12-614F | |||||
pen/strep/glutamine | GIBCO | 10378-016 | |||||
heparin | LEO | 012866-08 | |||||
Trypsin/EDTA (10x) | GIBCO | 15400-054 | |||||
Cells | |||||||
adipose deriverd MSCs | n.a. | n.a. | |||||
GFP-positive MSCs | n.a. | n.a. | |||||
human fibroblasts | n.a. | n.a. | |||||
143B cells | ATCC | CRL-8303 | |||||
FLUC-143B cells | ATCC | CRL-8303 | Transduced | ||||
Disposables | |||||||
Culture flasks 175 cm2 | CELLSTAR | 660175 | |||||
50 mL tubes | Greiner bio-one | 210261 | |||||
Freeze tubes | Thermoscientific | 377224 | |||||
Ultra-Clear tubes | Beckman | 344058 | Referred to in the manuscript as ultra-centrifuge tubes | ||||
0,22 µm filter | Millex | SLGV033RS | |||||
200 mesh Formvar-carbon-coated nickel grids | EMS (Electron Microscopy Sciences) | ||||||
0.5 mL insulin syringes with 29G Needle | Terumo | U-100 | |||||
Petri dish | Sigma – Aldrich | P7612 | |||||
Filter paper | Thermo fisher Scientific | 50363215 | |||||
Reagents / kits | |||||||
paraformaldehyde | Alfa Aeser | 43368.9M | |||||
PBS | Braun | 220/12257974/110 | |||||
glutaraldehyde | EMS (Electron Microscopy Sciences) | 16300 | |||||
uranyl oxalate | EMS (Electron Microscopy Sciences) | 22510 | |||||
urany acetate | EMS (Electron Microscopy Sciences) | 22400 | |||||
methyl cellulose | EMS (Electron Microscopy Sciences) | 1560 | |||||
PKH67 | Sigma | mini67-1kt | Referred to in the manuscript as GFLD | ||||
BSA | Sigma | A8412 | |||||
CBA – human inflammatory cytokine kit | BD | 551811 | |||||
Formaldehyde 37% | VWR | 104003100 | |||||
Carbon Steel surgical blades | Swann-Morton | 206 | Referred to in the manuscript as surgical knife | ||||
anti-human vimentin antibody | Santa Cruz | sc-6260 | Clone V9 | ||||
Antibody diluent | DAKO | S0809 | |||||
HRP-labeled anti mouse IgG antibody | Life Technologies | 32230 | |||||
DAB-kit | DAKO | K500711 | |||||
hematoxyllin | Sigma | GHS232 | |||||
EDTA-buffer | n.a. | n.a. | |||||
Citrate buffer | n.a. | n.a. | |||||
rabbit polyclonal anti-GFP antibody | Abcam | n.a. | Ab290 | ||||
DAPI | Life Technologies | D1306 | |||||
Paracetamol, 120 mg / 5 ml syrup | Bayer | n.a. | Sinaspril, paracetamol solution for kids | ||||
Isoflurane 1000 mg/g | Vumc pharmacy | n.a. | |||||
buprenofine hydrochloride, 0.3 mg/ml | Indivior UK Limited | n.a. | |||||
lidocaine-HCL 2% | Vumc pharmacy | n.a. | |||||
70% ethanol | VWR | 93003.1006 | |||||
Tissue glue | Derma+Flex, formulated medical cyanoacrylate | Vygon | LB604060 | ||||
Eyedrops: Vidisec Carbogel, 2 mg/ml | Bausch+Lomb | n.a. | |||||
D-luciferin, potassium salt | Gold Biotechnology | LUCK-1 | |||||
Glass slides | Thermo scientific | 630-0954 | |||||
Stainless steel loops | n.a. | n.a. | |||||
Mice experiments | |||||||
Mice, Hsd:Athymic Nude-Foxn1nu, female, 6 weeks at arrival, bacterial status conform FELASA | ENVIGO | n.a. | |||||
Paper-pulp smart home (cage enrichment) | Bio Services | n.a. | |||||
Alpha-dri bedding material | Shepperd Speciality Papers | n.a. | |||||
Mouse food: Teklad global 18% protein rodent diet | ENVIGO | 2918-11416M | |||||
Sutures | Ethicon | V926H | |||||
Scissors | Sigma-Aldrich | S3146-1EA | (or similar) | ||||
Tweezers | Sigma-Aldrich | F4142-1EA | (or similar) |