Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma-Associated Leptomeningeal Disease
March 29th, 2024
This article describes a protocol for the propagation of cerebral spinal fluid-circulating tumor cells (CSF-CTCs) collected from patients with melanoma-associated leptomeningeal disease (M-LMD) to develop preclinical models to study M-LMD.
Leptomeningeal disease from melanoma, or M-LMD, is an aggressive form of metastasis of the meninges where circulating tumor cells, or CTCs, infiltrate into the cerebral spinal fluid. Sadly, there's no effective treatments for M-LMD so understanding its biology will help develop therapies to reduce mortality. Recent reports have shown how CTCs can colonize the nutrient scarce CSF environment, such as utilizing the iron scavenging ability to enhance their survival and complement C3 to promote the invasion into the cerebral spinal fluid via the choroid plexus.
Therefore, CTCs are very good tools to study M-LMD. The biggest challenge in studying CSF CTCs is the overall lack of preclinical model and the scarcity of these cells. Finding ways to grow these rare and fragile cells from patients and generating a xenograft model will greatly benefit researchers in understanding M-LMD pathology and designing rational therapies.
We have developed a method which successfully propagated CSF CTCs from M-LMD patients in vitro and in vivo, which provided us the ability to perform molecular and functional analyses of M-LMD cells, as well as efficacy study in xenograft models. Though the current methodologies is imperfect, but being able to culture and expand CSF CTCs from melanoma patients for the first time, gave us insights on the importance of understanding the CSF microenvironment. To begin pre-coat a T175 flask with poly-l-lysine.
Place the flask in an incubator at 37 degrees Celsius for one hour. Now use a sterile serological pipette to aspirate the lysine solution. Pipette 30 milliliters complete meningeal culture medium containing human meningeal cells into the flask.
Incubate the cells at 37 degrees Celsius under 5%carbon dioxide supplementation. When cells reach approximately 75%to 80%confluency, collect and save the HMC cultured media in a 50 milliliter conical tube. Then add complete meningeal culture medium to the tube in a one-to-one ratio.
Finally, pipette the growth factors into the tube to create the HMC conditioned media. To process the cerebral spinal fluid, first, place it in a 15 milliliter conical tube on ice to cool it. Centrifuge the fluid in a pre-cool centrifuge at 257 x g for five minutes at four degrees Celsius.
Pipette out the supernatant without disturbing the cell pellet and make aliquots of it in different tubes. Now add one milliliter of PBS to the cell pellet to resuspend it. Centrifuge the cells at 1, 500 x g for five minutes at four degrees Celsius.
Aspirate the PBS supernatant while leaving about 50 microliters of it at the bottom of the tube. Perform a cell count with the cell suspension before culturing the cells. Several growth factors were upregulated in the media cultured with human meningeal cells.
To begin, spin the thawed cultures of cryopreserved CSF CTCs at 257 x g for five minutes at four degrees Celsius. Aspirate the supernatant with a pipette. Add one milliliter of PBS to wash the cell pellet, aspirate the supernatant again.
Next, resuspend the cells in 450 microliters of HMC conditioned media in three separate wells of a 96-well plate. Pipette 150 microliters of the cell suspension into three different wells of a 96-well plate. Top up the HMC conditioned media with fresh media every three days.
When the cell culture reaches 90%confluency, transfer the trypsinized cells of a well into an empty well in a 24-well plate. During the continued culture of the tumor cells, select the clones of the cells that transform and expand exponentially for further experimentation. To begin, identify the female immunodeficient NSG mice having leptomeningeal disease or LMD.
Place an anesthetized mouse on the surgical table. Position the nose of the mouse in a modified L-shaped cone of a stereotactic apparatus, ensuring that the nostrils stay unobstructed. Pulling the skin gently forward across the ventral surfaces of both pinnae.
Then fix it to the nose cone with tape. Guide the surgical scissor tips downward from the pinnae across the occipital bone. Create a small midline incision measuring five to seven millimeters just above the palpated concavity.
With a pair of blunt tipped forceps having one to two millimeter tips, gently apply pressure on the cisterna magna. Introduce the tips in a closed position and open them while exerting downward pressure on the dura. Continue to perform blunt dissection until the dural membrane is clearly discernible and the associated blood vessels are visible within the exposed area.
Now keep the forceps open to retract the surrounding musculature. Then attach a 27 to 29-gauge needle to a one milliliter syringe. Insert the syringe beneath the dura to visualize the bevel.
Gradually retract the syringe plunger to collect as much cerebral spinal fluid as possible. Transfer the fluid into a micro centrifuge tube immediately. Place it on ice.
Centrifuge the sample at 257 x g for five minutes at four degrees Celsius. After aspirating the supernatant, pipette 500 microliters of sterile PBS to the cell pellet. Centrifuge the pellet at 257 x g for five minutes at room temperature.
Discard the supernatant, then transfer the pellet to a 96-well plate containing HMC conditioned media.
Overview
This article describes a protocol for the propagation of cerebral spinal fluid-circulating tumor cells (CSF-CTCs) collected from patients with melanoma-associated leptomeningeal disease (M-LMD) to develop preclinical models to study M-LMD.
Key Study Components
Area of Science
- Neuroscience
- Oncology
- Cell Biology
Background
- Leptomeningeal disease from melanoma (M-LMD) is a severe form of metastasis.
- CTCs infiltrate the cerebral spinal fluid, complicating treatment options.
- Understanding CTC biology is crucial for developing effective therapies.
- Current methodologies for studying CSF CTCs are limited.
Purpose of Study
- To propagate CSF CTCs from M-LMD patients.
- To generate preclinical models for studying M-LMD pathology.
- To facilitate molecular and functional analyses of M-LMD cells.
Methods Used
- Pre-coating T175 flasks with poly-l-lysine for cell culture.
- Using human meningeal cells to create conditioned media.
- Centrifugation and processing of cerebral spinal fluid samples.
- Establishing xenograft models in immunodeficient mice.
Main Results
- Successful propagation of CSF CTCs in vitro and in vivo.
- Insights gained into the CSF microenvironment's role in CTC survival.
- Identification of growth factors that enhance CTC culture.
- Development of a methodology for studying M-LMD effectively.
Conclusions
- Propagation of CSF CTCs provides a valuable tool for M-LMD research.
- Understanding the biology of CTCs can lead to new therapeutic strategies.
- Further refinement of methodologies is needed for optimal results.
Frequently Asked Questions
Executive Industry Relevance
Establishing ex vivo cultures of CSF-derived circulating tumor cells (CTCs) from melanoma-associated leptomeningeal disease (M-LMD) addresses a critical bottleneck in preclinical oncology research. This capability enables mechanistic de-risking and target validation for therapies aimed at a highly lethal metastatic niche with limited treatment options. The approach supports predictive confidence in early discovery and translational continuity for portfolio advancement in neuro-oncology.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of therapeutic hypotheses in a disease-relevant CSF microenvironment.
- Supports functional validation of targets implicated in CTC survival and colonization.
- Facilitates mechanistic de-risking by modeling the biology of M-LMD at the cellular level.
- Provides a platform for pathway-specific inhibitor testing in patient-derived cells.
Screening & Assay Development
- Establishes reproducible ex vivo systems for quantitative assessment of drug efficacy.
- Standardizes culture conditions using HMC-conditioned media to enhance assay reliability.
- Enables expansion of rare CTCs for downstream molecular and functional analyses.
- Prepares validated cell lines for scalable compound screening workflows.
Translational & Preclinical Research
- Generates murine xenograft models for preclinical evaluation of candidate therapies.
- Aligns in vitro findings with in vivo disease progression and therapeutic response.
- Supports identification of translational biomarkers linked to CTC survival pathways.
- Enables risk-adjusted advancement of novel inhibitors targeting IGF and MAPK pathways.
Pipeline & Workflow Integration
This protocol bridges early discovery and preclinical validation by enabling the propagation and functional study of patient-derived CSF-CTCs. It integrates into workflows spanning target validation, assay development, and in vivo efficacy testing.
- Discovery Biology: Provides a platform for hypothesis testing and pathway elucidation in a clinically relevant context.
- Screening: Delivers reproducible, quantitative outputs for compound evaluation against patient-derived CTCs.
- Analytics: Supports molecular and cytokine profiling to compare survival factors and drug responses.
- Translational Research: Facilitates continuity from ex vivo culture to in vivo modeling and biomarker discovery.
- Enterprise Reuse: Establishes a reusable workflow for studying CTC-driven metastasis in neuro-oncology portfolios.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in M-LMD research.
- Operational Value: Standardizes rare cell culture and expands access to functional preclinical models.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient prioritization of neuro-oncology assets.
- Portfolio Impact: Supports risk-adjusted advancement and cross-program learning in metastatic disease research.
Implementation Considerations
- Requires expertise in rare cell isolation and ex vivo culture techniques.
- Demands access to specialized cell culture infrastructure and conditioned media preparation.
- Necessitates cross-team standardization for reproducibility and data comparability.
- May require adaptation for other tumor types or CSF-derived cell populations.
- Dependent on availability of patient-derived CSF samples and immunodeficient animal models.
Biopharma FAQs
Chapters in this video
0:00
Introduction
1:36
Preparation of Human Meningeal Cell (HMC) Conditioned Media and Collection of Cerebrospinal Fluid for the Culture of Circulating Tumor Cells
3:23
In Vitro Culture and Expansion of Cerebro Spinal Fluid (CSF)-Circulating Tumor Cells (CTCs) for the Study of Leptomeningeal Disease
4:24
CSF Collection from Mice with Leptomeningeal Disease for Subsequent Clone Expansion
