Ex Vivo Model Systems of Cancer-Bone Cell Interactions

Abstract

Ex vivo bone organ cultures preserve cell-cell and cell-matrix interactions and allow the study of cancer and bone cells in their natural tumor environment. This chapter is a comprehensive guide detailing the methods employed to establish and characterize ex vivo bone-cancer organ cultures. The chapter begins by outlining the materials and step-by-step procedures for successfully initiating and maintaining these cultures, emphasizing the advantage of modeling cancer within the bone microenvironment. Additionally, the chapter provides a detailed account of the techniques utilized to analyze key endpoints, such as tumor growth, microenvironmental changes, and therapeutic responses.

1. Introduction

Reproducing the complexity of the bone/bone marrow tumor niche and the dynamic interactions among bone cells and cancer cells in vitro remains a major challenge for biologists. Although osteoblastic and osteoclastic cell lines of rodent or human origin exist, they do not exhibit all the features of authentic bone cells. In addition, isolating primary cells from bone requires extensive digestion of extracellular matrix proteins and separation from other nonmesenchymal populations. This process and the subsequent in vitro culture deprive primary bone cells of their native spatial surroundings and lead to cellular phenotype and morphology changes, thus rendering cells not entirely representative of authentic bone cells. In addition, once placed in culture, primary cells frequently lose expression of characteristic genes or de-differentiate. Attempts to co-culture cancer and bone cells (either cell lines or primary cells) in 3D matrices have been only partially successful because of the lack of reproducible results.

Ex vivo bone organ cultures represent a departure from conventional in vitro cell cultures and introduce a sophisticated platform that preserves the native three-dimensional architecture and microenvironment of bone tissues, providing bone researchers with a dynamic and highly relevant tool for their investigations [1–8]. Ex vivo bone organ cultures involve the meticulous isolation of intact bone specimens, which are then cultured under carefully controlled conditions outside the living organism. This unique approach retains the intricate interplay of cells, extracellular matrix, and signaling molecules, offering a more nuanced representation of the in vivo bone microenvironment. The preserved physiological context enables researchers to explore a plethora of biological processes, including cellular interactions, bone remodeling, and the response to various external stimuli, mirroring the complexity of the living skeletal system. This methodology has proven invaluable in studying diseases affecting the skeletal system, such as osteoporosis, osteoarthritis, or cancer, providing a platform to investigate the molecular and cellular pathophysiological changes that occur at the tissue level [1–8]. In addition, ex vivo bone organ cultures are a versatile tool for testing and validating novel anti-tumor therapeutic strategies. Recreating the native bone environment enhances the translational potential of findings, facilitating a smoother transition from in vitro laboratory research to animals and, ultimately, to clinical applications.

In this chapter, we describe the procedures optimized by our group to establish ex vivo bone-cancer organ cultures using murine and human bones. Additionally, we describe different approaches applied to analyze various relevant endpoints in this culture system.

2. Materials

All ex vivo bone organ culture procedure steps should be performed under sterile conditions, and solutions, surgical tools, and culture tubes/dishes must be sterile.

1. Mouse calvarial and long bones: Full mouse calvarial or long bones (tibia/femur) can be isolated surgically from 8-day-old to 5-month-old male or female mice from wild-type or genetically modified mouse strains.

2. Human bones: Human bone pieces can be extracted from the femoral heads of healthy patients post-arthroplasty.

3. Dulbecco’s Modified Eagle Medium (DMEM-FBS): Add 10% fetal bovine serum (FBS, heat-inactivated) to DMEM with high glucose (Gibco, Catalog number: 11995065) and supplement with 100 U/mL penicillin +100 μg/mL streptomycin (Sigma-Aldrich, Co).

4. Tissue culture plastic materials: Individual cell culture dishes with a diameter of 100 mm (10 cm), 96- and 24-well culture plates (Fisher Scientific, Co.), 50 mL Falcon tubes, and 0.5- and 1.5-mL Eppendorf tubes (USA Scientific, Co.).

5. Cell culture incubator: Incubator at 37 °C with 5% CO₂ for cell cultures and mouse and human bone organ cultures.

6. Trypsin-EDTA: 0.25% trypsin-EDTA (Gibco Life Technologies).

7. Tissue fixative: Formalin 10% neutral buffered (Sigma-Aldrich, Co.).

8. Cancer cell lines: Human and murine cancer cell lines (i.e., myeloma and breast cancer).

9. Surgical tools: Surgical tools for dissection, forceps, scissors, scalpel, knife, etc.

10. Biopsy punch: Calvarial bone biopsy punch (Integra, Catalog number: 33–35).

11. Syringes: 1 mL syringe (Becton, Dickinson, and Company; 309,569) equipped with a 27G needle (Becton, Dickinson, and Company; 305,109).

3. Methods

3.1. Establishment of Calvarial Bone Organ Culture with Cancer Cells

1. Isolate the calvarial bones and remove the muscle and blood using sterile paper towels. Place the calvaria in a 24-well plate filled with 1 mL of sterile, cold PBS (see Fig. 1a).

2. Punch two holes in each parietal bone using a 5 mm biopsy punch, avoiding cranial sutures (see Fig. 1b).

3. Transfer the 5 mm calvaria disks, concave side up, to a 96-well plate.

4. Prepare a cell suspension of 50,000 cancer cells per 50 μL media. We use 50 μL of the cell suspension to ensure cancer cells are in close contact with the bone tissue and remain on top of the bone without overflowing. Transfer bone disks to a 96-well plate, placing their concave sides upward to enhance cancer cell retention.

5. Gently add 50 μL (50,000 cancer cells) of the cell suspension to the center of each calvarial disk using a pipette. Incubate this setup at 37 °C for 24 h. Adding the cell suspension immediately after isolating the bone disks is preferred to prevent the disks from drying out.

6. The following day, transfer the calvarial disks with cancer cells to a new 96-well plate. This step is essential to discard cancer cells that did not attach or engraft into the bone tissue, which typically accumulate at the edges of the wells without direct interaction with the calvarial disks. To transfer the calvarial disks, use forceps placed near the edge of the disks.

7. Fill the wells with 200 μL of media and incubate at 37 °C with 5% CO₂. Replace half of the media every 48 h. We recommend to maintain these cultures for up to 12 days.

8. Collection of the conditioned media periodically is advised to evaluate soluble factors released into the cell culture media (see Subheading 3.7.1). Harvest the calvarial disks for RNA/protein isolation and/or histological examination at the final time point.

Fig. 1.

(a) Step-by-step graphical depiction of the procedures for establishing ex vivo bone organ culture of the calvaria using either cancer cells or their conditioned media. 1. Obtain 6-days-old to 1-month-old mice. 2. Isolate calvarial disks using a punch biopsy tool and remove the soft tissue and debris. 3. Transfer calvarial disks to a 24-well plate with ice-cold PBS. 4. Transfer each calvarial disk to an independent well in a 96-well plate and start treatment with conditioned media or add cancer cells to establish cancer-bone organ cultures. 5. At the end of the study, (a) perform bioluminescence imaging or ELISA for tumor burden (if using cancer cells); (b) use the calvarial disks and conditioned for endpoint analyses (see Subheading 3.7) or freeze them at −80-degrees for future analyses. (b) Representative image of calvarial disks isolated using bone biopsy punch for ex vivo culture

3.2. Establishment of Calvarial Bone Organ Culture Treated with Cancer Cell Conditioned Media

1. As described in Subheading 3.1, start by isolating the calvaria, eliminating muscle and blood, and then placing the calvaria in a 24-well plate filled with sterile, cold PBS (see Fig. 1).

2. Repeat step 2 as outlined in Subheading 3.1.

3. Transfer 5 mm calvaria disks into a 96-well plate.

4. Subsequently, add 200 μL of a conditioned media solution to each well, ensuring that the calvarial disks are immersed in the media. We recommend using 50% conditioned media derived from cancer cells mixed with 50% complete media containing FBS. However, testing different concentrations of conditioned media is recommended for each experiment/cancer cell line.

5. Incubate this setup at 37 °C with 5% CO₂. Add the conditioned media solution immediately after transferring the disks to prevent the disks from drying out.

6. Replace the mixed media containing conditioned media every 72 h. We recommend maintaining these cultures for up to 12 days.

7. Follow the steps described in Subheading 3.1, step 8, to end the culture and assess endpoints.

3.3. Establishment of Mouse Long Bone Organ Cultures Treated with Conditioned Media from Cancer Cells

1. Isolate the long bones (tibias or femurs). Remove any surrounding muscle and blood and scrape off the periosteum. Place the collected bones in a Petri dish with sterile, cold PBS. Continue this process until all the bones are collected.

2. Next, carefully remove the metaphysis from both ends of the bones, as close to the growth plate as possible, while ensuring that the bone marrow remains intact and does not spill out. This step is crucial to effectively allow the media to flow through the bone samples.

3. Transfer these bones to a 24-well culture plate so they lie flat at the base of the well.

4. Prepare a conditioned media solution by mixing 50% cancer cell conditioned media with 50% complete media containing FBS. Add 500 μL of this conditioned media solution to each well, such that the bones are fully immersed in the media. We recommend using a 1:1 ratio of conditioned media and complete media. However, users may modify this ratio based on their specific experimental protocol. It is recommended to add the conditioned-media solution immediately after transferring the bones to prevent them from drying out.

5. Incubate this setup at 37 °C with 5% CO₂.

6. Replace the mixed media containing conditioned media every 72 h. We recommend maintaining these cultures for up to 12 days.

7. Follow the steps described in Subheading 3.1, step 8, to end the culture and assess endpoints.

3.4. Establishment of Mouse Long Bone Organ Cultures Inoculated with Cancer Cells

1. Isolate tibias or femurs, remove surrounding muscle and blood, and scrape off the periosteum. Place the collected bones in a Petri dish with sterile, cold PBS. Repeat this process until all the bones are collected (see Fig. 2).

2. Prepare a cell suspension containing 100,000 cancer cells per 20 μL. The number of cancer cells injected can be adjusted based on the aggressiveness of the cells being used. We use 20 μL of the cell suspension as this volume is small enough to be retained in the long bones without requiring the removal of bone marrow cells.

3. Load the cell suspension into a 1 mL syringe (Becton, Dickinson, and Company; 309,569) equipped with a 27G needle (Becton, Dickinson, and Company; 305,109). Inject 20 μL of the cell suspension into the bone through the metaphysis. For the tibia, inject through the proximal end, and for the femur, inject through the distal metaphysis until the needle tip is inside the bone marrow. Initially, you may encounter some resistance, which will gradually subside during injection.

4. After injecting, hold the needle in its place for a few seconds and then slowly withdraw it.

5. Place the injected bones in 0.5 mL tubes with the bone oriented upright and the injected end facing upward. Immediately immerse the bone in 300 μL of media containing 10% FBS to prevent the bone from drying. The media volume can be adjusted to ensure that the bone is fully immersed in the media. Create holes in their lids using a needle to facilitate air circulation inside the tubes.

6. Transfer the tubes to a rack and incubate them at 37 °C with 5% CO₂ for 24 h.

7. After 24 h, remove the bones from the tubes with forceps and carefully trim the metaphysis from the end where the cells were injected. Be cautious and remove only enough bone material to allow media to enter the bone without spilling out the marrow.

8. Transfer these bones to another set of tubes prepared in the same way as described in step 5.

9. Replace the mixed media containing conditioned media every 72 h. We recommend maintaining these cultures for up to 12 days.

10. Follow the steps described in Subheading 3.1, step 8, to end the culture and assess endpoints.

Fig. 2.

(a) Step-by-step depiction of the procedures for establishing ex vivo bone organ culture of the long bones (tibia or femur) using cancer cells. 1. Obtain 6-day-old to 3-month-old mice. 2. Isolate the tibia or femur and remove the soft tissue and debris. 3. Transfer the bones to a Petri dish with ice-cold PBS. 4. Inject 20ul of cancer cell suspension into the bone through its metaphysis using a syringe. 5. Place the injected bones in 0.5 mL tubes filled with media containing FBS. 6. At the end of the study, (a) perform bioluminescence imaging or ELISA for tumor burden; (b) use the bones and conditioned for endpoint analyses (see Subheading 3.7) or freeze them at −80-degrees for future analyses. (b) Representative image of tibia and femur isolated from 2-month-old mouse for ex vivo culture

3.5. Establishment of Human Bone Organ Cultures Treated with Conditioned Media from Cancer Cells

1. Ex vivo bone organ cultures can be performed using human bones, thereby bridging the gap between animal research and human health. These cultures can be performed using bone explants obtained from the femoral heads of healthy patients who have undergone arthroplasty. Working with human bone samples requires meticulous adherence to sterile procedures to prevent contamination and ensure accurate results. Researchers should be properly trained to work with human specimens. They should work in a sterile bio-safety level II fume hood and wear appropriate personal protective equipment (P.P.E.), including gloves, lab coats, glasses, and N95 masks to maintain a safe and controlled environment. Additionally, strict compliance with ethical guidelines, such as obtaining informed consent, de-identifying samples, and following institutional protocols (IRB, IBC), is required.

2. We recommend using specialized surgical tools to extract cylindrical pieces of bone tissue from the femoral head. Extract as much bone from the femoral head as possible and collect the bone pieces in a Petri dish containing DMEM and 10%FBS (see Fig. 3b).

3. Use a knife and forceps to cut the bone into equal-sized pieces.

4. Transfer the bone pieces to a 50 mL conical containing 15 mL PBS and vortex vigorously for 30 s.

5. Transfer the bone pieces to a new conical containing PBS and repeat step 4 three times. This will help to get rid of the blood and debris.

6. Transfer bones to a 24-well culture plate.

7. Prepare conditioned media solution by mixing 50% cancer cell conditioned media with 50% complete media containing FBS (DMEM +10% FBS). Add 500 μL of this conditioned media solution to each well, such that the bones are fully immersed in the media. Adjust the volume and ratio of the cancer cell conditioned media and complete media based on specific experimental needs. It is recommended to add the conditioned media solution immediately after transferring the bones to prevent the bones from drying out.

8. Incubate this setup at 37 °C with 5% CO₂.

9. Replace the mixed media containing conditioned media every 72 h. We recommend maintaining these cultures for up to 12 days.

10. Follow the steps described in Subheading 3.1, step 8, to end the culture and assess endpoints.

Fig. 3.

(a) Step-by-step representation of the procedures for establishing ex vivo organ culture of bone from the human femoral head using cancer cells. 1. Obtain the femoral head from a healthy patient. 2. Use specialized surgical tools (trocars) to extract cylindrical pieces of bone tissue from the femoral head and transfer them to a Petri dish with media containing FBS. 3. Transfer the bones to a 50 mL conical with PBS and vortex to eliminate the debris and blood. 4. Transfer the bones to 1.5 mL tubes and gently pipette the cancer cell suspension onto them. 5. At the end of the study, (a) perform bioluminescence imaging or ELISA for tumor burden; (b) use the bones and conditioned for endpoint analyses or freeze them at −80 degrees for future analyses. (b) Representative images of a human femoral head after arthroplasty and the cylindrical bone pieces extracted from the femoral head for bone explants

3.6. Establishment of Human Bone Organ Cultures Infiltrated with Cancer Cells

1. Collect human bones as described in steps 1–5 in Subheading 3.6.

2. Prepare a cell suspension containing 100,000 cancer cells per 150 μL. The number of cancer cells can be adjusted based on the aggressiveness of the cells, and the volume of media can be altered depending on the size of the bone pieces (see Fig. 3).

3. Use 1.5 mL tubes and perforate their lids using a needle. These holes will permit the circulation of air inside the tubes. Place the bones in these tubes using forceps and gently pipette 150 μL of the cell suspension onto the bone samples. Ensure that the bone is fully immersed in the cell suspension.

4. Transfer the tubes to a rack and incubate this setup at 37 °C with 5% CO₂.

5. Every 3 h, gently mix the cell suspension and pipette it back onto the bone. This will prevent the cells from settling down and allow maximal engraftment to the bone samples.

6. After 24 h, transfer the bones to a 96- or 24-well plate, depending on the bone size, using forceps. This step will help discard the cancer cells that did not infiltrate the bone and accumulated at the bottom of the tubes.

7. Fill the wells with media containing FBS., ensuring that the bone samples are completely immersed in the media.

8. Incubate this setup at 37 °C with 5% CO₂.

9. Replace the mixed media containing conditioned media every 72 h. We recommend maintaining these cultures for up to 12 days.

10. Follow the steps described in Subheading 3.1, step 8, to end the culture and assess endpoints.

3.7. Endpoints

3.7.1. Tumor Burden

The ex vivo tumor-bone organ culture system allows the quantification of tumor burden using various methods, including bioluminescence imaging, tumor biomarker analysis, and histological examinations. These methods provide quantification of tumor growth and progression and play a crucial role in assessing the efficacy of treatments.

Bioluminescence imaging (BLI) is a highly sensitive, noninvasive method for tracking tumor progression in small animal models. This technique often employs cells expressing firefly luciferase, an oxygenase that catalyzes the oxidation of D-Luciferin in the presence of molecular oxygen, requiring magnesium ions (Mg₂+) and adenosine triphosphate as cofactors. Luminescent photons emitted in this reaction can be captured by a highly sensitivity camera, enabling the visualization and localization of tumor cells within small animals [9]. The quantification of these photon emissions is a potent method for measuring tumor load and evaluating the effectiveness of various treatments. As outlined previously, luciferase-expressing tumor cells can be engrafted or injected into ex vivo bone organ cultures derived from either mice or humans and subjected to various treatments (see Fig. 4b). Similar to luciferase-expressing cells, cancer cells expressing a fluorescently tagged protein can also be utilized to detect and quantify tumor burden in these cultures (see Fig. 4a). Tumor burden can be measured by following the manufacturer provided instructions applicable to in vitro cultures (PerkinElmer, Catalog Number #122799). We use 150 μg/mL working solution of D-Luciferin diluted in PBS for human and mouse ex vivo bone cultures. The media is first aspirated from the ex vivo cultures, and then the bones are washed with PBS once. Subsequently, we add D-Luciferin-PBS to the media and incubate the bones for 10 min, protected from light. The bioluminescence is then assessed using an IVIS lumina XRMS system (Perkin Elmer, MA, US).

Fig. 4.

Ex vivo cancer-bone organ cultures with mouse and human bones. (a) Representative image of ex vivo calvarial bone organ culture with GFP+ myeloma cells. Yellow arrows indicate the GFP+ myeloma cancer cells. (b) Representative bioluminescence image analysis of mouse (tibia) and human bone (from femoral head post-arthroplasty) after 5 days of luciferase-positive breast cancer cell engraftment

For tumors that produce paraproteins, such as in multiple myeloma, the levels of these proteins can be assessed in the conditioned media from these cultures using ELISA (Enzyme-Linked Immunosorbent Assay) [10–14]. We recommend diluting the condition media 1:1 with the dilution buffer of these ELISA kits.

3.7.2. Bone Remodeling

The ex vivo bone organ culture systems are highly representative of the in vivo bone microenvironment and can be utilized for evaluating bone formation and resorption by measuring specific secreted bone biomarkers or histological quantification of osteoblast or osteoclast numbers (see Subheading 3.7.3). Our group has previously demonstrated that in ex vivo cancer-bone organ cultures, myeloma cells induce bone resorption, leading to a rapid increase in the circulating levels of the bone resorption marker C-telopeptide of type 1 collagen (CTX) while concurrently decreasing the levels of the bone formation marker type I procollagen N-terminal propeptide (P1NP) in the collected conditioned media [10–12, 14]. These results mirror the changes observed in animal models and patients. We recommend initially analyzing a few samples to determine whether dilution of the conditioned media is necessary for obtaining valid results before proceeding with the full experiment.

3.7.3. Secreted Factors

In addition to markers of bone formation or resorption, the conditioned media from bone organ cultures can be analyzed for levels of secreted factors related to tumor burden or bone metabolism using ELISAs or western blot approaches.

3.7.4. Gene Expression

Ex vivo bone organ cultures provide a valuable platform for investigating changes in gene expression patterns in response to pathological conditions and treatment interventions. Specifically, our group has used ex vivo multiple myeloma-bone organ cultures to examine the impact of cancer cells and different therapeutic agents on mRNA and protein gene expression [10–14].

3.7.5. Histological Sections

After the experiment is completed, ex vivo bone organ cultures can be processed for histological analysis to quantify osteoblast and osteoclast numbers, evaluate tumor burden, and perform immunostaining. An example of this approach is provided by Curtin et al., which utilized histological analyses of calvarial bone organ cultures to demonstrate that conditioned media from breast and prostate cancer cell lines induce osteoclastic bone resorption and increase the synthesis of PGE2 by bone [15].

4. Notes

1. While ex vivo bone organ cultures can be established with bones from mice at different ages, the reproducibility and the magnitude of the effects improve when using bones from younger mice (6 days old to 1 month old).

2. The calvarial and long bone tissues should be free of soft tissues. The soft tissue can be carefully trimmed or removed using a paper towel without causing any damage to the bones.

3. Maintaining ex vivo bone organ cultures with enriched media can improve cell viability when keeping the cultures for longer periods. An example of enriched cultured media for ex vivo bone organ cultures can be found here [13].

4. The use of mouse calvarial bone and human cancer cells in the model systems has the advantage of specifically visualizing and discriminating between the human cancer cells and those of the mouse bone cells using species-specific primers or antibodies.

5. Cultures of genetically modified mice (or human samples) may be established with bones of different sizes. We recommend recording bone weight before the experiment to correct protein measurements in the conditioned media.

6. This chapter did not cover the establishment of osteocyte-enriched ex vivo bone organ cultures. Protocols to establish such cultures are described here [1].

7. For human bone cultures, it is important to consider if the patient has skeletal issues or is taking medications that might influence bone and, therefore, the results of the experiment.

8. We do not recommend mixing different types of bones in the same experiment, as the expression of genes can differ between bone types.

9. To minimize the potential confounding effects of age, gender, and interpatient heterogeneity, we do not recommend using human bone samples from different patients in the same experiment.