Name: dual effects of melanoma cell-derived factors on bone marrow adipocytes differentiation
Uploaded: 2018-08-23T12:30:00
Description: Watch this Scientific Journal Video about Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation at JoVE.com

We thank Dov Zipori (The Weizmann Institute of Science, Rehovot, Israel) kindly for providing us the murine bone marrow stromal cell line 14F1.1. This study was supported by grants from the Chinese National Natural Science Foundation (Nos. 81771729) and the Yongchuan Hospital of Chongqing Medical University (Nos. YJQN201330; YJZQN201527).

NOTE: All cells used in this protocol should be grown for at least three generations after thawing from frozen stock cells.
1. Harvest Melanoma Cell-derived Factors
<ol>
	<li>Preparations
	<ol>
		<li>Obtain B16F10 cells and a mouse melanoma cell line. 
		NOTE: For this protocol, a mouse melanoma cell line was obtained from the Stem Cell Bank of the Chinese Academy of Sciences.</li>
		<li>Make a complete medium for the B16F10 cell culture (100 mL). Use Dulbecco&#39;s Mod...

<ol>
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Cocultures with inserts have been widely used to study cell-to-cell interactions. The 2D coculture system is an effective way to observe how the two parts crosstalk in vitro, by which we here showed two different cancer cell-driven effects on bone marrow adipocytes. Many labs have utilized this method to investigate the crosstalk between adipocytes and cancer cells6,12,27,39.
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Bone metastases are widespread among advanced cancer patients, but a curative treatment is still unavailable. Beyond specializing in storing energy as fat, adipocytes can support tumor growth and metastasis in bone marrow and other organs1,2,3,4,5,6. Moreover, adipocytes play an essential role in regulating cancer cell biology7,8,9,10 and metabolism4,11,12,13,14,15,16, as well as in bone metastasis1,4,12. In the bone marrow niche, adipocytes can also affect the biological behavior of cancer cells4,6,17. The interplay between bone marrow adipocytes and cancer cells with osteotropism is significant for an understanding of bone metastasis. However, little is known.
Based on the current studies, various methods are applied to adipocytes, including two- or three-dimensional (2/3D) and ex vivo cultures17,18,19,20,21. Recently, Herroon et al. designed a new 3D-culture approach to study interactions of bone marrow adipocytes with cancer cells22. Although the 3D coculture is optimal for mimicking physiological interactions between adipocytes and cancer cells in vivo, it suffers from poor reproducibility22,23. In comparison to a 2D coculture system, a 3D coculture system may provide different cellular phenotypes, such as cell morphology21,22,24,25,26. Moreover, the ex vivo culture of isolated cancellous bone tissue fragments can lead to a robust outgrowth of adipocytes from cultured bone marrow cells17.
In contrast to these previous models, however, the 2D cell culture model remains a classic, reliable, and easy technique for quickly scanning candidate molecules and the phenotypes changed in either adipocytes or cancer cells in vitro1,4,6,12,15,27. To better understand the crosstalk between bone marrow adipocytes and melanoma cells, we provide a detailed protocol for a 2D coculture system of bone marrow adipocytes with melanoma cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:684px;" width="685">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:18px;width:215px;">DMEM</td>
			<td style="width:140px;">Invitrogen Inc.</td>
			<td style="width:143px;">11965092</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Fetal Bovine Serum</td>
			<td style="width:140px;">Invitrogen Inc.</td>
			<td style="width:143px;">16000&#8211;044</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Phosphate Buffered Saline</td>
			<td style="width:140px;">Invitrogen Inc.</td>
			<td style="width:143px;">14190-144</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Insulin</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">91077C</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">3-isobutyl-1-methyl-xanthine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">I5879</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Dexamethasone</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">D4902</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Oil Red o</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">O0625</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">24-well plate</td>
			<td style="width:140px;">Corning</td>
			<td style="width:143px;">CLS3527</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Transwell insert</td>
			<td style="width:140px;">Millipore</td>
			<td style="width:143px;">MCHT24H48</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Penicillin/Streptomycin</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:143px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">isopropanol</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:143px;">I9516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">0.25% trypsin</td>
			<td style="width:140px;">Thermo Scientific</td>
			<td style="width:143px;">25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">hemocytometer</td>
			<td style="width:140px;">Bio-Rad</td>
			<td style="width:143px;">1450016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Culture incubator</td>
			<td style="width:140px;">Thermo Scientific</td>
			<td style="width:143px;"></td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">50ml falcon</td>
			<td style="width:140px;">Corning</td>
			<td style="width:143px;">CLS430828</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Clean Bench</td>
			<td style="width:140px;">Thermo Scientific</td>
			<td style="width:143px;">-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Microscopy</td>
			<td style="width:140px;">Olympus</td>
			<td style="width:143px;">-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">200 &#956;L pipet tips</td>
			<td style="width:140px;">BeyoGold</td>
			<td style="width:143px;">FTIP620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">1000 mL pipet tips</td>
			<td style="width:140px;">BeyoGold</td>
			<td style="width:143px;">FTIP628</td>
			<td></td>
		</tr>
	</tbody>
</table>

dual effects of melanoma cell-derived factors on bone marrow adipocytes differentiation

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are available for studying the significant crosstalk; however, a two-dimensional transwell system for coculture remains a classic, reliable, and easy way for this crosstalk study. Here, we present a detailed protocol that shows the coculture of bone marrow adipocytes and melanoma cells. Nevertheless, such a coculture system could not only contribute to the study of cell signal transductions of cancer cells induced by bone marrow adipocytes, but also to the future mechanistic study of bone metastasis which may reveal new therapeutic targets for bone metastasis.

In the bone marrow, adipocytes can appear in the tumor microenvironment1,13,33,34,35 at an early stage for supporting tumor progression through soluble factors or activating osteoclastogenesis6,12,36, especially in the context of obesi...

Watch this Scientific Journal Video about Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation at JoVE.com

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

This method can help answer key questions in the bone metastasis research field, such as what changes are induced in the bone marrow adipocyte growth and their development in a cancer microenvironment. The big advantages of this technique are that it is easy to perform and that the candidate molecules can be quickly obtained. This method can provide insights into the crosstalk between bone marrow adipocytes and the melanomal cells.It can also be applied to other cell types, such as osteoblasts. Begin by seeding two times 10 to the fifth B16F10 melanoma cells in a 100-millimeter dish with 10 milliliters of DMEM supplemented with 10%FBS, for a 24-hour incubation at 37 degrees Celsius, and 5%CO2. The next day, wash the about 80%confluent cell culture two times with five milliliters of PBS per wash, and starve the cells with 10 milliliters of serum-free culture medium in the cell culture incubator for 18 to 24 hours.The next day, use a pipette to transfer the conditioned supernatant into a 50-milliliter conical tube for centrifugation, and load the medium into a syringe equipped with a 0.45 micron-pore filter. Then, strain the solution into a sterile container to remove any remaining cell debris, and store the conditioned medium at minus 20 degrees Celsius for up to one month. To induce bone marrow adipocytes, culture mouse F14 1.1 bone marrow stromal cells in a 60-millimeter dish with complete DMEM to an 80%confluency.At the appropriate stage of culture, harvest the cells with 1.5 milliliters of a 0.25%trypsin solution for one to two minutes at 37 degrees Celsius. When the cells have detached, arrest the enzymatic reaction with five milliliters of medium supplemented with 10%FBS, and adjust the cells to a one to two times 10 to the fifth cells-per-milliliter concentration with fresh serum-supplemented culture medium. Seed five times 10 to the fourth cells in 0.5 milliliters of adipogenic induction medium per well in a 24-well plate, and return the cells to the cell culture incubator for another two days, until they reach about 90%confluency.Then, replace the supernatant with one milliliter of adipogenic maintenance medium per well, and continue to culture the cells until adipocyte maturation. When the cells look like small to large soap bubbles under light microscopy, wash these mature adipocytes two times with PBS, followed by fixation in 0.2 milliliters of 3.7%formaldehyde for one hour. Rinse the fixed cells in 60%isopropanol, and allow the wells to dry completely.Add 0.2 milliliters of Oil Red O working solution to the cells for a 30 to 120-minute incubation at room temperature, and wash the cells with water to remove any unbound dye for imaging of the adipocytes by light microscopy. Alternatively, upon their maturation, add 0.5 milliliters of ready-to-use RNA isolation reagent to each adipocyte culture well, and use the appropriate primers to confirm the adipocyte-specific gene signature of the cells. Then, run the reaction in a thermal cycler using the indicated settings.To set up a bone marrow-derived adipocyte melanoma cell co-culture, first add 150 microliters of serum-free DMEM to the inner chambers of 0.4 micron-pore size membrane inserts, and place each insert into one well of a 24-well plate containing 500 microliters of serum-free medium per well. Dilute B16F10 melanoma cells from an 80%cell culture to an experimentally-appropriate concentration in fresh serum-free medium, and replace the medium in the inner chamber of each insert with 150 microliters of cells. Next, replace the adipogenic maintenance medium supernatant in each well of a 90%confluent mature bone marrow adipocyte culture with 600 microliters of complete DMEM, and transfer the melanoma cell-containing inserts to each well of the mature adipocyte culture plate.When all of the inserts have been transferred, place the plate in the cell culture incubator. After two days, remove the inserts and wash the bottom of each well two times with 500 microliters of PBS per wash. Then, stain the adipocytes with the Oil Red O working solution, or analyze the adipocyte gene expression by quantitative PCR, as demonstrated.Mature adipocytes differentiated in tumor-conditioned medium contain lipid droplets, allowing the differentiated adipocytes to be easily identified by Oil Red O staining. The adipocyte-specific gene signature of these cells can also be confirmed by quantitative PCR. A reduction in lipid storage is observed in adipocytes co-cultured in the presence of high numbers of melanoma cells, as well as a de-differentiation in adipocyte phenotype, as evidenced by a decreased adipocyte-specific gene expression in these formerly mature cells.After watching this video, you should have a good understanding of how to design and set up a 2D co-culture system for studying the interactions between two different cell types.

dual-effects-melanoma-cell-derived-factors-on-bone-marrow-adipocytes

Research

JoVE Journal

Cancer Research

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed to mechanical stress due to hemodynamic flow. One of these stresses that circulating tumor cells (CTCs) experience is fluid shear stress (FSS). While cancer cells may experience low levels of FSS within the tumor due to interstitial flow, CTCs are exposed, without extracellular matrix attachment, to much greater levels of FSS. Physiologically, FSS ranges over 3-4 orders of magnitude, with low levels present in lymphatics (&#60;1 dyne/cm2) and the highest levels present briefly as cells pass through the heart and around heart valves (&#62;500 dynes/cm2). There are a few in vitro models designed to model different ranges of physiological shear stress over various time frames. This paper describes a model to investigate the consequences of brief (millisecond) pulses of high-level FSS on cancer cell biology using a simple syringe and needle system.

Here we demonstrate a method to apply fluid shear stress to cancer cells in suspension to model the effects of hemodynamic stress on circulating tumor cells.

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

Murine Model for Studying Leptomeningeal Disease Using Circulating Tumor Cells

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

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.

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

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 ...

Murine Bone Marrow Harvest and Isolation for the Characterization of Myeloid Leukemia Microenvironment 

To begin, remove the tissue from the bones of the euthanized dissected mouse. Place the cleaned bones into a new six-well plate containing FACS buffer placed on ice. Transfer the bones into a mortar with two to five milliliters of FACS buffer.Grind the bones in a circular motion until the bone marrow is released. With a three milliliter syringe, pull up and flush down the bone marrow to homogenize it. Pull the homogenate into another syringe, then filter it through a 70 micrometer cell strainer into a 50 milliliter tube.Rinse the tissue chunks from their filter back into the mortar with FACS buffer before homogenizing a second time. Filter the rinsed tissue as before. Next, rinse the remaining bone spicules back into the mortar with FACS buffer.Then flush the pieces into a 15 milliliter tube to ensure maximum yield. To digest the bone marrow, first centrifuge it at 300 G for five minutes at four degrees Celsius. Resuspend the bone marrow pellet in two milliliters of digestion mixture.Transfer the mixture to a 15 milliliter tube. Then incubate it at 37 degrees Celsius for 45 minutes on a rotator. Now, add 10 milliliters of FACS buffer to stop the enzymatic digestion.Filter the mixture through a 70 micrometer cell strainer into a 50 milliliter tube. Centrifuge the suspension at 400 G for seven minutes at four degrees Celsius. Resuspend the pellet in one milliliter of RBC lysis buffer.Next, add 10 milliliters of FACS buffer to halt the lysis. Then filter the mixture as before into a 50 milliliter tube. Lastly, centrifuge the mixture at 300 G for five minutes at four degrees celsius.After discarding the supernatant, resuspend the pellet in 100 microliters of FACS buffer. For the digestion of bone spicules, first, vortex the bone spicules. Decant the supernatant and retain the bones at the bottom.Next, resuspend the spicules in one milliliter of the bone spicule digestion mixture. Incubate the tube on a rotator for 60 minutes at 37 degrees Celsius. Finally, stop the digestion with 10 milliliters of FACS buffer.Filter the mixture into the 50 milliliter tube containing RBC lysed and digested bone marrow.

Murine Bone Marrow Staining for the Characterization of Myeloid Leukemia Microenvironment

To begin, mix the isolated murine bone marrow suspension with the bone spicule suspension. Pipette 10 microliters of the cell suspension onto a hemocytometer for counting. Centrifuge the cell suspension at 300 G for five minutes at four degrees Celsius.Then resuspend the pellet in 100 microliters of FACS buffer. To stain the cells with antibodies for magnetic depletion, add FC block, CD45 APC, and TER-119 APC. Then wash the cell suspension with FACS buffer.After centrifuging the remaining mixture, resuspend the pellet in 100 microliters of FACS buffer. To stain the cell suspension with microbeads for magnetic depletion, add mouse IgG and anti-APC microbeads. Incubate the mixture on ice for 20 minutes.Wash the LD column with two milliliters of MACS buffer. Resuspend the cells in the buffer, then filter it through a five milliliter test tube with a 35 micrometer cell strainer cap. Next, set the LD column on the magnetic separator stand and place a five milliliter test tube under the column to collect the eluted material.Pipette the cell suspension into the LD column and collect the negative fraction flow through in the test tube. Wash the column two times with one milliliter of MACS buffer and collect the eluent material in the same tube. Place the LD column over a new five milliliter test tube.With a pipette, dispense three milliliters of buffer into the column, then flush out the positively labeled cells into a five milliliter test tube. Centrifuge both the positive and negative fractions, then resuspend the pellet in 100 microliters of FACS buffer. To stain the CD45 TER-119 negative fraction add one microliter of each antibody for every 10 to the sixth cells.Place the suspension on ice for 20 minutes. Then wash the suspension with two milliliters of FACS buffer before centrifuging. Add one milliliter of the buffer to the pellet and pipette propidium iodide for live cell staining.Finally, filter the sample through a 35 micrometer cell strainer into a five milliliter test tube before analyzing the cells on a multicolor flow cytometer. Arteriolar endothelial cells significantly expanded in the acute myeloid leukemia microenvironment with a concomitant loss in the sinusoidal endothelial populations. A small expansion in the mesenchymal stromal cells were seen at eight weeks of age.