Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines
September 13th, 2017
This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.
The overall goal of this in vitro human preclinical model of osteoclastogenesis derived from peripheral blood monocytes, or PBMCs, cultured with breast cancer cell lines is to mimic cancer cell osteoclast interactions. This method can help to answer key questions in the field of bone metastasis about the effects of cancer cell and bone cell interaction within the bone microenvironment. The main advantage of this technique is that it is a fully human pre-clinical model.
Also, this method can provide insight into bone metastasis. It can also be applied to the study of other bone-related pathological mechanisms, such as osteoporosis. Individuals new to this method may struggle because of the variability among healthy donors and in selecting the appropriate PBMC cell density.
Visual demonstration of this method is critical as they need see our PBMC selection and seeding steps require some manual experience before proficiency can be achieved. Begin by diluting at least 20 milliliters of healthy-donor whole blood in EDTA at a one to one ratio in PBS. After mixing thoroughly, split the blood solution into 30 milliliter aliquots in 50 milliliter conical tubes.
Carefully underlay 15 milliliters of lymphocyte separation medium into the bottom of each tube. Separate the cells by density gradient centrifugation. And pull the white mononuclear-cell containing buffy coats into a new 15 milliliter tube.
Wash the harvested cells two times in 20 milliliters of PBS per wash, followed by lysis of the red blood cells with five milliliters of red blood cell lysis buffer for three to five minutes, on ice. Stop the reaction with 20 milliliters of PBS, and collect the white blood cells by centrifugation. Resuspend the pellet and complete alpha M-E-M for counting.
Then, plate the cells at a seven point five times 10 to the five PBMC per square centimeter concentration in each well of a 24 well plate for their incubation at 37 degrees celsius and five percent CO2. After about three hours, remove the supernatant, debris, unattached cells, and unlysed erythrocytes from the cultures. And add fresh medium, supplemented with MCSF.
Fourteen days after seeding, wash the cells two times with PBS, and fix them in four percent paraformaldehyde for 20 minutes at room temperature. Perform trap staining according to the manufacturer's instructions. The osteoclast-like cells will be TRAP positive with at least four nuclei.
Count the osteoclast-like cells manually under the microscope at a 10X magnification. For cancer cell induced osteoclast differentiation when the cancer cells reach ninety-percent confluency, they're detached by trypsinization and seeded onto zero point four micrometer pore inserts at a four times 10 to the three cells per square centimeter concentration in fresh alpha MEM. The next day, place the seeded inserts over undifferentiated one-day plated mononuclear cell cultures in complete alpha M-E-M, and change the medium every two to three days.
Then, 11 days after the start of the co-culture, transfer the inserts into a new plate containing the appropriate reagent for the desired downstream analysis. Breast cancer cells can sustain osteoclastogenesis, as the number of osteoclasts in the wells induced by cancer cells is similar to that obtained in the positive growth-factor control wells. The number of osteoclasts observed in all of the cultures is reduced however, when the cells are treated with an anti-tumor drug.
Interestingly, the surface areas of matured osteoclasts derived from growth factors are larger than those induced by cancer cells. This effect is not observed in anti-tumor drug treated cultures. Once mastered, this technique can be completed in seven eight hours if it is performed properly.
Following this procedure, additional downstream analysis can be performed as gene-expression analysis, Western Blot, immunofluorescence analysis, or ELISA to answer additional questions about cancer-cell/bone-cell interactions or about anti-tumor drug mechanisms. After its development this technique pave the way for researchers in the field of bone metastasis to study the bone microenvironment, in fully human preclinical model. After watching this video you should have a good understanding of how to co-culture monocytes, undergo differentiation with cancer cells.
Don't forget that working with biological samples and drugs can be extremely hazardous and that precautions such as wearing gloves and lab coats should always be taken while performing this procedure.
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Overview
This protocol describes the development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. This model can help answer key questions regarding bone metastasis and improve therapeutic options.
Key Study Components
Area of Science
- Osteoclastogenesis
- Bone metastasis
- Cancer cell interactions
Background
- The model utilizes peripheral blood monocytes (PBMCs).
- It aims to replicate the interactions between cancer cells and osteoclasts.
- This research is significant for understanding bone-related pathologies.
- Variability among healthy donors can affect results.
Purpose of Study
- To mimic cancer cell-osteoclast interactions.
- To investigate the effects of these interactions within the bone microenvironment.
- To provide insights into bone metastasis and related conditions.
Methods Used
- Culture of PBMCs with breast cancer cell lines.
- Assessment of osteoclastogenesis.
- Evaluation of cancer cell and bone cell interactions.
- Analysis of variability in PBMC cell density.
Main Results
- The model successfully mimics cancer cell-osteoclast interactions.
- Insights gained can inform therapeutic strategies for bone metastasis.
- Potential applications extend to other bone-related diseases.
- Challenges include donor variability and cell density selection.
Conclusions
- This human preclinical model is valuable for studying bone metastasis.
- It enhances understanding of cancer cell interactions in the bone microenvironment.
- Future studies can leverage this model for broader applications in bone pathology.
Frequently Asked Questions
Executive Industry Relevance
This fully human preclinical model enables mechanistic de-risking of osteoclastogenic pathways in bone metastasis by recapitulating cancer cell-osteoclast crosstalk in a physiologically relevant system. It supports target validation and assay development for anti-tumor therapeutics by providing quantitative readouts of osteoclast differentiation and function. The model enhances predictive confidence in lead identification by allowing direct observation of reciprocal interactions and downstream analysis of both cell types under drug treatment conditions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses regarding tumor-induced osteoclastogenesis and pathway clarification in the bone microenvironment.
- Operational Value: Supports biological de-risking and functional target validation by modeling osteoclast differentiation from primary human monocytes under defined co-culture conditions.
- Scientific Value: Facilitates predictive confidence and portfolio triage by quantifying osteoclast formation in response to cancer cell stimuli versus growth factor controls.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream workflows through standardized osteoclast differentiation from PBMCs using RANKL and MCSF.
- Operational Value: Addresses assay standardization and reproducibility via fixed cell density seeding (7.5×10⁵ PBMC/cm²) and timed differentiation (14 days) with TRAP-based osteoclast quantification.
- Scientific Value: Enables reliable compound evaluation by testing drug effects on osteoclastogenesis in both indirect (conditioned medium) and direct co-culture formats.
Translational & Preclinical Research
- Scientific Value: Supports disease relevance modeling for osteoporosis and other bone conditions through adaptation of the osteoclastogenesis assay.
- Operational Value: Ensures translational continuity by using fully human cells, reducing species translation risk in preclinical-to-clinical extrapolation.
- Scientific Value: Enables risk-adjusted advancement decisions by measuring changes in osteoclast number and surface area as pharmacodynamic biomarkers of drug efficacy.
Pipeline & Workflow Integration
The model fits within the discovery continuum from target validation through lead identification to preclinical efficacy testing, particularly for bone-targeted therapies and bone microenvironment modulators.
- Discovery Biology: Supports hypothesis testing of cancer cell-derived factors in osteoclast activation and pathway clarification of RANKL-independent osteoclast induction mechanisms.
- Screening: Delivers assay readiness through standardized monocyte isolation, differentiation, and co-culture with breast cancer cell lines on permeable inserts for paracrine and juxtacrine signaling studies.
- Analytics: Provides quantitative osteoclast readouts via TRAP staining, multinucleated cell counting, and morphometric analysis (surface area) to compare experimental conditions.
- Translational Research: Connects to preclinical continuity by enabling study of drug effects on the metastatic bone microenvironment using clinically relevant breast cancer cell lines.
- Enterprise Reuse: Functions as a reusable platform for studying osteoclast modulation across multiple cancer types and bone pathologies beyond breast cancer.
Operational & Enterprise Impact
- Scientific Value: Delivers predictive confidence in target validation by reducing mechanistic ambiguity in osteoclast-cancer cell interactions.
- Operational Value: Ensures standardization and reproducibility through defined PBMC isolation, seeding density, and cytokine supplementation protocols.
- Strategic Value: Improves go/no-go decisions by enabling early assessment of drug effects on osteoclast formation and function, reducing late-stage biological risk in bone metastasis programs.
- Portfolio Impact: Supports risk-adjusted prioritization by quantifying osteoclastogenic potential of compounds and guiding advancement based on bone microenvironment modulation.
Implementation Considerations
- Requires expertise in primary human cell isolation, culture, and differentiation techniques, including PBMC separation and monocyte adherence.
- Depends on instrumentation for cell counting, centrifugation, microscopy (10X magnification for osteoclast counting), and incubation (37°C, 5% CO₂).
- Necessitates cross-team standardization of PBMC donor sourcing, cell density optimization (7.5×10⁵ PBMC/cm²), and co-culture timing (11 days post-cancer cell seeding) to minimize variability.
- Involves adaptation considerations when extending to non-breast cancer cell lines or alternative osteoclast stimuli beyond RANKL/MCSF.
- Includes practical limitations such as donor variability in osteoclast differentiation potential and the need for manual proficiency in PBMC seeding and co-culture setup.
