A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Summary

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.

Abstract

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.

Introduction

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 osteosarcoma^2,3,4,5,6,7. MSCs are multipotent stem cells that reside in various adult and fetal tissues, including bone marrow, adipose tissue, placenta, umbilical cord blood, and others^8,9. 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 stroma^2,3,11,12,13. 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 vivo^18,19, internalization by endothelial cells can stimulate tumor angiogenesis and increase vascular permeability^16,20, 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.

Protocol

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.

1. Prepare EV-depleted Fetal Bovine Serum (FBS) by centrifuging FBS at 70,000 x g overnight (16 hours), no brake, at 4 °C using an ultra-swinging bucket rotor (expect 1 h for deceleration). Carefully collect the supernatant (EV-depleted FBS) without disturbing the pellet. Filter the EV-depleted FBS with a 0.22 µm filter.
2. Prepare EV-depleted culture medium by supplementing Iscove's Modified Dulbecco's Medium (IMDM) with 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM glutamine (1x P/S/G) and 5% EV-depleted FBS.
3. Seed 3 x 10⁶ 143B cells in 175 cm² flasks and culture them in EV-depleted culture medium in an incubator at 37 °C and 5% CO₂. When the cells are 80 - 90% confluent (typically after 36 h to 48 h), collect the supernatant from 8x 175 cm² flasks (28 mL/flask) for EV-isolation.
4. Pre-clear the cell supernatant to remove cells and cell debris by differential centrifugation (centrifuging the supernatant of the previous spin each time) according to the following protocol:
   1. Centrifuge the supernatant twice at 500 x g for 10 min.
   2. Centrifuge the supernatant twice at 2000 x g for 15 min.
   3. Centrifuge the supernatant twice at 10,000 x g for 30 min.
   4. Perform all centrifugations at 4 °C with maximum acceleration and deceleration speed settings. After each step, carefully collect the EV-containing supernatant, leaving about 1 mL behind. Proceed immediately to step 1.5 or store the pre-cleared conditioned medium at -80 °C until EV isolation.
5. Isolate EVs by centrifuging the pre-cleared conditioned medium once at 70,000 x g for 1 h, slow brake, at 4 °C in ultra-centrifuge tubes (final volume 38.5 mL/tube) using an ultra-swinging bucket rotor.
6. Carefully remove the supernatant, leaving about 1 mL behind, resuspend the EV-containing pellets in the remaining volume, pool them all in one ultracentrifuge tube, and add phosphate-buffered saline (PBS) until complete tube filling (final volume 38.5 mL).
7. Centrifuge once again at 70,000 x g for 1 h at 4 °C, without brake (expect 1 h for deceleration). Carefully remove the supernatant without disturbing the pellet and leave 100 - 200 µL behind.
8. Resuspend the EV-pellet and adjust the final volume to 200 µL with PBS (standardized for all EV-isolations). Use the EV preparation immediately or store at -80 °C until use²².
   NOTE: EVs can be stored up to 6 months at -80 °C.

2. EV Characterization.

Purified EVs can be visualized by transmission electron microscopy (TEM)²³.

1. Mix EV preps with an equal volume of 4 % paraformaldehyde in phosphate buffer.
2. Coat 200 mesh Formvar-carbon-coated nickel TEM grids with 5 µL of EV suspension for 20 min at room temperature.
3. Fixate the EV samples on TEM grids with 1 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.0), contrast with uranyl oxalate (pH 7.0), and embed in a mixture of 4 % uranyl acetate and 2 % methyl cellulose in a 1:9 ratio on ice.
4. Remove grids with stainless steel loops and blot the excess fluid with filter paper to ensure an appropriate thickness of the methyl cellulose film.
5. After drying, examine grids with a transmission electron microscope and capture images with a digital camera coupled to the corresponding image software.

3. Education of MSCs by tumor-derived EVs.

1. Prepare MSC culture medium by supplementing alpha-minimum essential medium (alpha-MEM) with 4% EV-depleted human Platelet Lysate (hPL)²⁴, heparin (10 U/mL) and 1x P/S/G.
   NOTE: EV-depleted hPL is obtained following the procedure described in section 1.1.
2. Seed 1.4 x 10⁶ adipose-derived MSCs per 75 cm² flask and culture them in EV-depleted MSC-culture medium in an incubator at 37 °C and 5% CO2.
3. When cells reach 70% confluence, add 143B osteosarcoma- or control human fibroblasts (hf)-EVs, 10 µL EV preparation/cm² of culture flask. Incubate for 24 hours and add the same amount of EVs for additional 6 hours. NOTE: Human fibroblasts are cultured in Dulbecco's Modified Eagle Medium (DMEM) 10% FBS and EVs are isolated as described in section 1.
4. Collect the MSC supernatant, centrifuge 1x at 500 x g for 5 min at 4 °C (with maximum acceleration and deceleration speed settings), transfer to a new tube and store the cleared supernatant at -80 °C to enable future cytokine analysis (section 5).
5. For in vivo experiments (section 6), educate GFP-positive MSCs²⁵ as described in sections 3.1 to 3.3, collect the cells and resuspend them in PBS at a final concentration of 10⁶ cells per 100 µL. Keep cells on ice until injection.

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:

1. Briefly, add 180 µL of diluent to the 200 µL EV prep. Dilute 1 µL of GFLD dye in 50 µL of diluent, and add 20 µL of diluted dye to the diluted EV prep.
2. Gently mix by pipetting and incubate for 3 min at room temperature in the dark.
3. Add 5 mL of 0.1 % BSA in PBS, transfer to ultra-centrifuge tubes and fill the tube with PBS (final volume 38.5 mL/tube).
4. Centrifuge 1x at 70,000 x g for 1 h at 4 °C, without brake (expect 1 h for deceleration). Carefully remove the supernatant and resuspend the labeled EV-pellet in a final volume of 200 µL (adjust volume with PBS).
5. Add the labeled EVs to the MSC culture or store them at -80 °C. After overnight incubation, assess vesicle internalization by fluorescence microscopy and flow cytometry⁷.

5. Assessment of MSC Cytokine Expression Profile.

1. For the multiplex quantification of interleukin-8 (IL-8), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) protein levels in the MSC conditioned medium (see step 3.4), use a commercially available cytometric bead array (CBA) for human inflammatory cytokines following the manufacturer's instructions.
2. Briefly, mix the antibody-conjugated capture beads and add them to all assay tubes. Add the cytokine standard dilutions or the conditioned medium samples to the tubes.
3. Add the detection reagent and incubate 3 hours at RT.
4. Wash the beads with the supplied washing solution. Measure the samples using a flow cytometer and analyze the data according to the manufacturer's instructions.

6. Generation of an Orthotopic Xenograft Mouse Model of Osteosarcoma.

1. Allow six-week-old, female, Athymic Nude-Foxn1nu mice to acclimatize for at least one week before starting the experimental procedures.
2. One day before the surgical procedure and as peri-operative analgesia add paracetamol to the drinking water. Dilute "paracetamol solution for kids" (see Table of Materials) with water to get a final concentration of 2 mg/mL. Based on an average water intake of 150 mL/kg/day, and an average body weight of 20 g, this dose is sufficient to reach a dosage of 6 mg/mouse/day (300 mg/kg/day).
3. Continue analgesic treatment until 24 hours after the operation, or longer if the animals show signs of pain.
4. On the day of the surgical procedure, collect cultured luciferase-positive (Fluc) 143B cells (previously generated by lentiviral transduction) by centrifuging at 300 x g for 10 min and resuspend the cells at a concentration of 2 x 10⁵ cells/µL in PBS. Keep the cell suspension on ice up to 6 hours.
5. Autoclave surgical equipment. Prepare and clean the working area and place the sterilized scissors and tweezers on sterile sheets. Twenty min before the surgical procedure (step 6.8), inject the animals subcutaneously with buprenorphine (0.05 mg/kg, diluted in 0.9% saline) as analgesic using 0.5 mL insulin syringes (29-gauge needle).
6. Just before anesthetizing each animal, fill a 10 µL syringe with at least 1 µL of the concentrated (Fluc) 143B cell suspension (see step 6.4).
7. Anesthetize the animal with isoflurane inhalation anesthesia (2 - 3% in oxygen). Check the level of anesthesia by toe-pinching the animal. When the animal does not react to the pinching, i.e., it is deeply anesthetized, apply ointment on the eyes to prevent dryness during anesthesia.
8. Position the animal on its back with the head in an anesthesia mask and with the legs towards the operator. Flex the left knee. Wipe the skin with 70% ethanol and apply lidocaine (2%) with a cotton tip 3 times as local analgesic.
9. Use a sterile surgical knife to make a small incision (approximately 5 mm) on the skin just below the knee to expose the tibia. Drill a pinhole in the tibia, approximately 2 mm below the knee using a 0.8 mm micro twist drill. Be cautious not to drill through both tibia cortices.
10. Inject 1 µL cell suspension (approximately 2 x 10⁵ cells) slowly (in approximately 5 seconds) into the hole using a 26-gauge needle. Close the hole with a droplet of tissue glue to prevent backflow of the suspension.
11. Close the skin with monofilament sutures and one drop of tissue glue. Let the animal recover in a well-heated environment (use a heat lamp). Do not leave animals unattended until they have regained sufficient consciousness to maintain sternal recumbency. Only after the animals are fully recovered from anesthesia, return them to their own cages.
12. Two days after tumor inoculation, inject EV educated or naïve GFP-positive MSCs (10⁶ cells in 100 µL PBS, see step 3.5) intravenously with a 0.5 mL insulin syringe in the tail vein of the mice.
13. Follow tumor growth by bioluminescence imaging²⁶ twice a week. To this end, inject the mice i.p. with 150 µL D-luciferin (30 mg/mL) with an insulin syringe. Ten min after injection, position the animals in the bioluminescence imaging system and measure the photon-flux generated by tumor cells.
14. In addition, measure the diameter of the primary bone-tumor with a caliper. The tumor volume (in mm³) is estimated by width x length² x 0.5.
15. NOTE: Humane endpoints are defined as tumor diameter >15 mm or weight loss >15%.

7. Assessment of Lung Nodule Number

1. When one of the animals reaches the humane endpoint defined in step 6.14 (in approximately 3 - 4 weeks), terminate the experiment and collect and analyze all relevant tissues.
2. Ten min before euthanization, inject 150 µL D-luciferin (30 mg/mL) with a 0.5 mL insulin syringe.
3. Anesthetize the animals with isoflurane inhalation anesthesia (see step 6.7). Confirm the depth of anesthesia by the absence of reactions of the mouse to toe-pinching. Euthanize the mice by cervical dislocation²⁷.
4. Collect lungs, liver, spleen and kidneys using sterilized scissors and tweezers. Wash the organs in PBS to remove blood traces and put them on a Petri dish.
5. Place the Petri dish with the organs in the bioluminescence imaging system. Measure the bioluminescence signal on both sides of the organs. Immediately after measuring, plunge the organs in 4% formalin to fixate them  (24 - 72 hours, at RT) for future histological analysis.
6. Process the obtained pictures with appropriate imaging software by manual thresholding. Count the number of lung nodules on each picture. Count the total number of lung nodules on both sides of the lungs.

8. Histological Analysis

1. For histological analysis of the lung tissues:
   1. Embed the formalin-fixed organs in paraffin and prepare 6 µm tissue slides.
   2. Deparaffinate the lung tissue slides and perform antigen retrieval by boiling them for 15 min in 0.01 M citrate buffer (pH 6).
   3. Incubate the slides horizontally at room temperature with anti-human vimentin antibody (200 µg/mL) diluted 1:150 in antibody-diluent (commercially available) and subsequently with the secondary, HRP-labeled antibody (0.5 mg/mL, dilution 1:500). Stain the tissues with 3'-diaminobenzidine (DAB) and hematoxylin counter staining.
   4. Observe the stained tissue slides using an optical microscope coupled to the corresponding camera and image software.
2. To assess the presence of GFP-positive MSCs in mouse tibias:
   1. Decalcify the tibias in ethylenediaminetetraacetic acid (EDTA) 0.24 M, pH 7.2 - 7.4. Refresh EDTA buffer every other day until bones become flexible (approximately 1 week).
   2. Dehydrate tibias and infiltrate them with paraffin. Embed the tibias (including osteosarcoma tissue) in paraffin blocks.
   3. Use a microtome to prepare 6 µm paraffin sections. Place the paraffin sections onto glass slides. After drying, store slides overnight at room temperature.
   4. Perform heat-mediated antigen retrieval using citrate buffer (see 8.1.2) and stain the tissues with anti-GFP antibody (whole antiserum) in a 1:900 dilution in antibody-diluent. Counterstain the tissues with 4',6-diamidino-2-phenylindole (DAPI).
   5. Observe the tissue slides with a fluorescence microscope coupled to the corresponding imaging software.

Results

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

Discussion

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 activa...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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)