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 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.
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.
The collection of deidentified patient CSF specimens was approved by the University of South Florida's Institutional Review Board (IRB) (MCC 50103, 50172, and 19332). Patients with M-LMD may be diagnosed in several ways, including positive CSF cytology, a characteristic magnetic resonance imaging (MRI) of the brain and/or spine, or a combination of clinical findings with suggestive MRI findings. CSF from these M-LMD patients were collected routinely as a part of their standard clinical care. No procedure is performed unless there is a clinical indication. Informed consent was obtained from patients for sample collection and using them for research and publication. The generation of in vivo murine-LMD models was approved by the University of South Florida Institutional Animal Care and Use Committee (IACUC# IS00010398). The overall scheme of this protocol is summarized in Figure 1. The details of the reagents and equipment used in the study are listed in the Table of Materials.
1. Preparation of HMC-conditioned media
2. Collection of CSF and sample processing
3. In vitro culture and expansion of CSF-CTCs
4. In vivo inoculation of CSF-CTCs to generate cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) model
NOTE: A PDX model involves the engraftment of cancer cells directly from a cancer patient (without ex vivo culture), whereas the CDX model uses cancer cell lines or, in this case, CTCs that have been propagated and immortalized18.
5. CSF collection from mice with LMD for subsequent clone expansion
Understanding the requirements for successful CSF-CTCs growth ex vivo is an ongoing effort. To that end, it is believed that providing essential factors that mimic the CSF microenvironment is of key importance22. Human meningeal cells (HMCs) secrete a variety of growth factors into the CSF, including FGF-2, EGF, IGFBP2, and IGFBP6, and are known to support the growth of CTC cells12,13,14,23,24. Therefore, a human cytokine array analysis was performed on HMC-conditioned media to identify potentially important components required for CTC survival. Indeed, several growth factors were upregulated in the media cultured with HMCs (Figure 3A). For example, granulocyte-macrophage colony-stimulating factor (GM-CSF), VEGF-A, and IGFBPs (IGFBP2, 3, 4, and 6).
The CSF-cellular components from patients may consist of multiple cell types, such as CTCs, immune cells, and fibroblasts. Non-CTCs will eventually stop passaging overtime. Generally, cells that propagate successfully and remain in proliferation are cancer (M-LMD) cells. Validation of growing cells in culture is indeed M-LMD cells, which can be done by IF detection of MLANA expression and transcriptomic analyses, which have previously been shown7.
As a proof of concept to show the potential use and application of established in vitro and in vivo PD-CSF-CTC lines, single-cell RNA-sequencing (scRNA-seq) analysis was used, and the results revealed several genes that were enriched and retained from the uncultured patient CSF-CTCs7. Two of them include receptor tyrosine-protein kinase ErbB3 and IGF-1R, which have implications on melanoma progression and chemotherapy resistance25,26,27.
To test whether they played a role in CSF-CTC survival, a crystal violet proliferation assay was conducted on PD-CSF-CTCs treated with FDA-approved drugs tucatinib and ceritinib that target ErbB28 and IGF-1R7,29 respectively. Anti-IGFBP2 antibody was included as a positive control that should hinder the growth of PD-CSF-CTC cultures. The results showed that the absence of IGFBP2 or IGF-1R was effective in reducing the proliferation of PD-CSF-CTCs (Figure 3B). Given that MAPK signaling is downstream of IGF-1R, calcein-AM live cell staining and MTT cell survival assays were also performed in three M-LMD PD-CSF-CTC lines by treating them with ceritinib or the MAPK inhibitors, dabrafenib and trametinib or a combination of all three. The data demonstrated that the viability of all three cell lines was significantly reduced by ceritinib, whereas dabrafenib and trametinib had mixed effects (Figure 3C). The result from debrafenib and trametinib treatments was surprising. All three PD-CSF-CTC lines were derived from M-LMD patients that harbored a BRAFV600E mutation7. This may suggest an acquired chemo-resistance effect of CSF-CTCs, which is something to be investigated in the future.
Next, as an example of how PD-CSF-CTCs can be utilized in vivo, murine-M-LMD models were established by intrathecally inoculated with varying numbers of PD-CSF-CTCs. The median survival times in mice were determined (Figure 3D). To visualize M-LMD progression, PD-CSF-CTC lines were tagged with a bioluminescent marker, such as the NL reporter system21, and captured by BLI (Figure 2C). The location of the LMD metastases was also demonstrated using immunohistochemistry with protein melan-A (MLANA)30 as a marker of the melanoma cells (Figure 3E). As a proof of concept to test therapeutic strategies against M-LMD in vivo, murine-M-LMD cohorts were given daily oral monotherapy of ceritinib or trametinib, or a combination of dabrafenib and trametinib or ceritinib and trametinib. The control (untreated) cohort received oral saline as a comparison. The results showed a significantly prolonged survival (Figure 3F) and delayed disease detection (Figure 3G) in the cohort that was treated with ceritinib and trametinib (untreated M-LMD median survival: 28.5 days vs. ceritinib and trametinib treated M-LMD median survival: 38.5 days; P value = 0.0052). These data underscore the potential usefulness of the developed M-LMD PD-CSF-CTC lines for conducting preclinical studies to determine the efficacy of novel therapeutics.
Figure 1: A schematic overview of the process of establishing patient-derived CSF-circulating tumor cells (PD-CSF-CTCs). CSF from patients can be sampled via lumbar puncture, Ommaya reservoir, or rapid autopsies. Through a series of in vitro and in vivo propagations, each step generates an intermediate CSF-CTC culture (i.e., patient CSF-CTCs, in vitro culture, in vivo culture) until establishing a PD-CSF-CTC line. Please click here to view a larger version of this figure.
Figure 2: Examples of in vitro and in vivo culturing of CSF-CTCs derived from M-LMD patients. (A) Representative brightfield images showing the in vitro growth of an M-LMD CSF-CTC colony at 6 weeks and 9 weeks in HMC-conditioned media. Scale bar: 1000 µm. (B) MRI images at 4 weeks and 8 weeks after intrathecally inoculated with PD-CSF-CTCs; a successful establishment of a murine model of M-LMD. Yellow arrows point to enlarged ventricles and possible hydrocephaly in this M-LMD mouse. (C) Representative BLI visualization of M-LMD development in mice. The figure is adapted from Law et al.7. Please click here to view a larger version of this figure.
Figure 3: PD-CSF-CTC lines are used in various preclinical experiments to study M-LMD. (A) A human cytokine array showing an increase of different secreted growth factors (i.e., IGFBPs, VEGF-A, and GM-CSF) in culture media (MenCM) in the presence of human meningeal cells (HMCs). (B) A scanned image of a crystal violet cell proliferation assay to determine the efficacy of anti-IGFBP2 antibody, tucatinib, and ceritinib against one of the PD-CSF-CTC lines. The control condition was given vehicle treatment. The experiment was performed in triplicate. (C) Cell survival assay of three different established PD-CSF-CTC lines (from patients 09, 12, and 16) in vitro. Cells were treated with either ceritinib (cer), a combination of dabrafenib (dab) + trametinib (tra), cer + tra, or all three. Cells were collected at 72 h after treatment. Calcein-AM staining was used to visualize cell viability, and an MTT assay was used to determine cell survival. A paired sample t-test was used for statistical analysis. Scale bars: 20 µm. (D) A survival curve of a murine M-LMD model. NSG mice were inoculated intrathecally (via the cisterna magna) with one of the PD-CSF-CTC lines at 10,000, 20,000, and 50,000 cells. The median survival of M-LMD mice was determined. (E) IHC detection for MLANA, a marker for melanoma, in the brain sections of M-LMD mice. Positive MLANA was found in the meninges (stained in red; pointed by yellow arrows), whereas the normal (healthy) brain did not show cancer growth (negative for MLANA). Scale bars: 200 µm. (F) A representative efficacy experiment of murine M-LMD cohorts given either daily oral saline, cer, tra, dab/tra or cer/tra. Survival of mice was determined. The log-rank (Mantel-Cox) test was used for statistical analysis. (G) Representative BLI images of M-LMD progression in 5 weeks, comparing control (saline) treated vs. cer/tra treated murine M-LMD cohorts. Panel (C) of the figure is adapted from Law et al.7. Please click here to view a larger version of this figure.
Table 1: Summary of clinical CSF-CTCs obtained for ex vivo culture in M-LMD patients. A summary table of 11 M-LMD patients, which their CSF-CTCs have been attempted to propagate. The patients in the Table were previously characterized in Law et al.7. Please click here to download this Table.
M-LMD is a devastating, universally fatal disease, and there is an urgent need to find better treatment strategies. One of the major barriers to studying M-LMD is the inability to acquire enough CSF-CTCs to perform molecular and functional studies1,7. Though there are existing methods to culture CTCs from peripheral blood and CSF of other cancer types, such as breast and ovarian cancers11,31,32, these CTC propagation methods are usually short-term, and there has been no reported success in culturing CSF-CTCs from melanoma. In addition, the current methodologies for propagating CTCs exist in short-term ex vivo settings and have yet to yield an in vivo LMD model derived from patient LMD cells. Here, a novel protocol is presented to culture these cells in vitro and in vivo, leading to unique patient-derived cell lines. Currently, of 11 M-LMD patients in the study, there was an approximately 60% (7 of 11) chance of success in propagating M-LMD CSF-CTCs in vitro, and while this was lowered to ~20% (2 of 11) in vivo using the CDX method7.
It is clear that in vitro conditions do not recapitulate the CSF microenvironment. However, proteomic approaches have previously been performed to study protein components in the CSF and provided some insights as to key factors that were required for CTC growth ex vivo3. For example, it was identified that one of the major pathways promoting CTC survival in M-LMD patients was associated with heightened IGF-related activities3,7. Further, studies have shown that the leptomeninges secretes a variety of cytokines/growth factors into the CSF, including FGF-2, EGF, GM-CSF, and proteins related to IGF-signaling12. Indeed, this was recapitulated in the media cultured with HMCs, supporting a potential role for these growth factors in promoting CSF-CTC growth.
A major advantage in generating a PDX (or CDX) model is the ability to gain deeper insights into the pathology of disease, something that in vitro conditions lack. Ideally, a PDX approach is preferred since the CSF-CTCs would be directly from patients without ex vivo culturing. Initially, attempts were made to create M-LMD using this approach, but they have not been successful thus far. The difficulty in generating PDX mice is possibly associated with the abundance and integrity of the starting material (i.e., very few viable CTCs in patient CSF at routine collection in the clinic). This may explain why we had superior success growing CTCs from CSF collected at autopsy7. To increase the probability of in vivo propagation, this protocol was modified to provide an alternate CDX approach. CSF-CTCs can be first expanded in vitro (step 3) to generate PD-CSF-CTC lines that have long-term and greater growth potential. These cells are then inoculated in mice to create M-LMD. Though the current method generated a limited number of in vivo CDX M-LMD (~ 20%) models, this might reflect the transcriptional diversity of CSF-CTCs, the complexity of the CSF microenvironment, and the difficulty in culturing these cells in general. We posit that future development of a humanized mouse model may enhance the engraftment success rate given the importance of the microenvironment in supporting cancer cell viability33.
A limitation of the CDX approach is that only certain clones were selected from patient samples, and genetic drift of cancer cells through ex vivo culturing may no longer reflect the transcriptional profile of the original source. However, it has been reported that despite in vitro culturing, PD-CSF-CTC lines retained approximately 97% similarity of gene expression to isolated, non-cultured patient CSF-CTCs7. In that study, scRNA-seq analyses revealed overlapping enriched gene signatures between non-cultured, in vitro PD-CSF-CTCs and in vivo PD-CSF-CTCs, such as SOX9, ErbB3, and IGF-1R7, suggesting these may be potential therapeutic targets. Additionally, these commonly enriched genes are involved in biological pathways associated with transcriptional regulation and metabolism7. Collectively, this highlights the translational value of PD-CSF-CTC cultures for better understanding the biology of M-LMD, identifying targetable molecular mechanisms and pathways driving the disease, and designing rational therapies in future studies.
Though the current methodology remains imperfect, as there is no way to predetermine the status and viability of CSF-CTCs in M-LMD patients, several observations have been made that would increase the likelihood of success since the CTCs are low in number and quite fragile. These critical steps include coordinating with the clinic to have CSF samples placed on ice as soon as they are drawn and have them quickly transported to the lab so as to maintain cellular integrity. Subsequently, CSF-CTCs should be processed immediately, either by plating them in culture or cryopreserving the cells.
Overall, culturing and expanding CSF-CTCs was a trial-and-error process, but the establishment of this protocol to generate patient-derived M-LMD cells will give researchers the resources required to perform experiments with patient samples, which could not have been done previously. A major goal moving forward is to utilize M-LMD PD-CSF-CTCs to conduct molecular characterization, high throughput drug screening, and in vivo drug efficacy studies to design rational therapies to treat M-LMD. It is believed that this approach will lead to treatment strategies that will greatly reduce the morbidity and mortality associated with this currently fatal aspect of advanced metastatic melanoma.
The authors have nothing to disclose.
We would like to thank patients and families for their extraordinary generosity in donating tissue for this scientific study. This work was supported by grants from the National Institutes of Health grants P50 CA168536, R21 CA256289, R21 CA216756 (to KSMS and PAF) K99 CA226679 (to IS). Moffitt Foundation Research Acceleration Fund (to BC and PAF), Moffitt Chemical Biology & Molecular Medicine Program (PAF and DD), Moffitt Foundation (PAF). The Molecular Genomics, Tissue, and Bioinformatics & Biostatistics Shared Resource Cores at Moffitt is supported in part by the National Cancer Institute through a Cancer Center Support Grant (P30-CA076292) and the Moffitt Foundation.
1 mL syringe 27 – 29 G needles | Any vendor | n/a | 0.1 mm Sterile Filtered |
1.5 mL Eppendorf tubes | Any vendor | ||
15 ml and 50 mL polystyrene centrifuge tubes | Any vendor | n/a | |
6 - 8 weeks females NOD SCID gamma (NSG) mice | Charles River | Males optional | |
Buprenorphine Sustained-Release (Bup-SR) | Zoopharm | DEA controlled | |
Fetal bovine serum (FBS) | ScienCell | #0010 | |
Gas inhalation anestehsia system | VeteEquip | #901812 | COMPAC5 |
Hamilton microliter syringes | Hamilton | 10, 25, 50, and 100ml | 30 G for cisterna magna injection |
Human basic fibroblast growth factor (bFGF) | Milipore Sigma (or any vender) | #F0291 | |
Human epidermal growth factor (EGF) | Milipore Sigma (or any vender) | #SRP3027 | |
Human meningeal cells (HMCs) isolated from human leptomeninges | ScienCell | #1400 | |
IVIS 200 imaging system | Caliper Life Sciences | n/a | |
Magnifying glass with light | Any vendor | n/a | |
Meningeal Cell Culture Media (MenCM) | ScienCell | #1401 | |
Meningeal cell growth supplement (MCGS) | ScienCell | #1452 | |
MRI imaging | Bruker | BioSpec series | Optional |
P1000, P200, P20 pipettes/ pipette tips | |||
penicillin-streptomycin Antibiotic solution | ScienCell (or any vender) | #0503 | |
Phosphate buffered saline (PBS) | Any vendor | n/a | 0.1 mm Sterile Filtered |
Rodent Surgical Instruments (Scissors, Forceps) | Roboz Surgical Instrument (or any vendor) | ||
Screw cap cryo tubes | |||
Sterile blue paper/ drape covering | Any vendor | n/a | n/a |
Sterile cotton sticks | Any vendor | n/a | |
Tissue culture plates/flasks (96-well, 24-well, 12-well, 6-well, T175 etc.) |
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