preparation of human meningeal cell &#40;hmc&#41; conditioned media and collection of cerebrospinal fluid for the culture of circulating tumor cells

Murine Model for Studying Leptomeningeal Disease Using Circulating Tumor Cells

Leptomeningeal disease (LMD) occurs when circulating tumor cells (CTCs) disseminate into the cerebral spinal fluid (CSF) and establish in the meninges, the membrane surrounding the brain and spinal cord1,2. LMD can occur in several cancers but is particularly prevalent in melanoma. In advanced stages of melanoma, approximately 5% of patients will develop melanoma-associated M-LMD2,3. While relatively low in regard to other sites of metastasis, the consequences of M-LMD are devastating, with overall survival ranging from weeks to months, and is a significant contributor to patient morbidity1,3,4. This is primarily due to a paucity of effective treatment options combined with major gaps in our knowledge regarding how the leptomeninges are colonized by melanoma cells2. Therefore, understanding the biology of M-LMD will facilitate in designing novel therapies to improve clinical outcomes.
Recent reports have shown how CTCs colonize the unique CSF microenvironment. For example, Complement C3 promotes the invasion of tumor cells into the CSF via the choroid plexus, an intricate network of blood vessels in each ventricle of the brain5. Further, in response to the scarce micronutrients in the CSF, CTCs can upregulate lipocalin-2, an iron-scavenging protein, and its receptor SLC22A17 to enhance survival6. Using omic-based analyses of CSF, our group also found that the CSF is enriched with proteins that regulate insulin-like growth factor (IGF) signaling, as well as innate immunity3. Together, these data emphasize the value of CSF-CTCs from liquid biopsies to study M-LMD.
While CSF-CTCs can sometimes be identified by sampling patient CSF via lumbar puncture, Ommaya reservoir, or rapid autopsies, a major limitation is obtaining sufficient numbers of these rare and fragile cells1,7. For example, using the CTC enumeration technique, only several hundred to several thousand tumor cells are identifiable per patient CSF sample7, which makes it difficult to perform molecular and functional analyses in vitro or in vivo. Though there have been reports of success in briefly growing CTCs ex vivo from peripheral blood (i.e., breast cancer CTCs)8,9,10, these cells usually only grow for the short term, and there have been no reported cases of being able to grow melanoma CTCs in the CSF. Hence, finding ways to propagate melanoma CSF-CTCs, or CTCs in general, will be highly beneficial to study the biology of M-LMD7,11.
For the first time, a novel technique to propagate CSF-CTCs from M-LMD patients ex vivo is described. Here in this report, a detailed protocol was developed that allows for the culture and expansion of CSF-CTCs from M-LMD patients. Since the meninges secrete a variety of growth factors such as FGF, IGF, VEGF-A, and IGFBPs that support the growth surrounding its environment12,13,14,15,16, it was rationalized that CSF-CTCs may require these components to grow in ex vivo conditions. Therefore, this protocol uses conditioned media generated by culturing human meningeal cells- (HMCs-) in vitro. For in vivo inoculation, patient-derived cells are inoculated into immunodeficient mice to generate patient-derived CSF-CTCs (PD-CSF-CTCs) lines. The availability of patient-derived M-LMD cells will support cellular, molecular, and functional assays to study M-LMD and propose novel treatment strategies for this deadly disease.

Author Spotlight: Assessing the Potential of Circulating Tumor Cells in Leptomeningeal Disease Research

Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma&#45;Associated Leptomeningeal Disease

Leptomeningeal disease from melanoma or MLMD 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 MLMD, so understanding its biology will help develop therapies to reduce mortality. Recent reports have shown how CTCs can colonize the nutrient scar 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 MLMD. 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 MLMD pathology and designing rational therapies.We have developed a method which successfully propagated CSF CTCs from MLMD patients in-vitro and in-vivo, which provided us the ability to perform molecular and functional analyses of MLMD 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 Microenvironments.

Preparation of Human Meningeal Cell &#40;HMC&#41; Conditioned Media and Collection of Cerebrospinal Fluid for the Culture of Circulating Tumor Cells

To begin, precoat 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-cooled centrifuge at 257 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 resususpend it.Centrifuge the cells at 1, 500 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.

preparation-human-meningeal-cell-hmc-conditioned-media-collection

Research

JoVE Journal

Cancer Research

61 Views. To begin, precoat 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 con...

Video: Preparation of Human Meningeal Cell (HMC) Conditioned Media and Collection of Cerebrospinal Fluid for the Culture of Circulating Tumor Cells

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the meninges, the membrane layers that protect the brain and the spinal cord. Once established, the prognosis for M-LMD patients is dismal, with overall survival ranging from weeks to months. This is primarily due to a paucity in our understanding of the disease and, as a consequence, the availability of effective treatment options. Defining the underlying biology of M-LMD will significantly improve the ability to adapt available therapies for M-LMD treatment or design novel inhibitors for this universally fatal disease. A major barrier, however, lies in obtaining sufficient quantities of CTCs from the patient-derived CSF (CSF-CTCs) to conduct preclinical experiments, such as molecular characterization, functional analysis, and in vivo efficacy studies. Culturing CSF-CTCs ex vivo has also proven to be challenging. To address this, a novel protocol for the culture of patient-derived M-LMD CSF-CTCs ex vivo and in vivo is developed. The incorporation of conditioned media produced by human meningeal cells (HMCs) is found to be critical to the procedure. Cytokine array analysis reveals that factors produced by HMCs, such as insulin-like growth factor-binding proteins (IGFBPs) and vascular endothelial growth factor-A (VEGF-A), are important in supporting CSF-CTC survival ex vivo. Here, the usefulness of the isolated patient-derived CSF-CTC lines is demonstrated in determining the efficacy of inhibitors that target the insulin-like growth factor (IGF) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, the ability to intrathecally inoculate these cells in vivo to establish murine models of M-LMD that can be employed for preclinical testing of approved or novel therapies is shown. These tools can help unravel the underlying biology driving CSF-CTC establishment in the meninges and identify novel therapies to reduce the morbidity and mortality associated with M-LMD.

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.

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the ...

In Vitro Culture and Expansion of Cerebro Spinal Fluid &#40;CSF&#41;&#45;Circulating Tumor Cells &#40;CTCs&#41; for the Study of Leptomeningeal Disease

To begin, spin the thawed cultures of cryopreserved CSF-CTCs at 257 G for five minutes at four degrees Celsius. Aspirate the supernatant with the 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.

CSF Collection from Mice with Leptomeningeal Disease for Subsequent Clone Expansion

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 cerebrospinal fluid as possible. Transfer the fluid into a microcentrifuge tube. Immediately place it on ice.Centrifuge the sample at 257 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 g for five minutes at room temperature.Discard the supernatant, then transfer the pellet to a 96-well plate containing HMC conditioned media.