This work was supported by the government of the Principality of Monaco, as well as by &#39;Le Groupement des Entreprises Mon&#233;gasques dans la Lutte contre le cancer&#39; (GEMLUC) and Flavien Foundation, which provided the means for BD FACS Melody purchase.

The present study was conducted using DAOY wild-type (WT) medulloblastoma cell lines, which were cultured at 37 &#176;C with 5% CO2 in DMEM medium supplemented with 8% FBS. The xCT-deleted cell line was maintained under the same conditions, with experiments carried out in media supplemented with 1 mM N-acetylcysteine (NAC). The cells were regularly screened for mycoplasma using a commercially available Mycoplasma Detection Kit (see Table of Materials) and were cultured up to the 10th passage.
1. Harvesting and seeding the cells
NOTE: All steps are performed using sterile aseptic techniques in a tissue culture laminar flow hood. DAOY medulloblastoma cells are adherent, meaning all unattached cells can be discarded by washing with phosphate-buffered saline (PBS) without Ca2+ and Mg2+.
<ol>
	<li>Take the stock dish (Petri dish, 100 mm diameter) with the cells and remove the floating cells and medium from the plate using an aspirator.</li>
	<li>Add 5-10 mL of PBS to the dish, wash the bottom with circular movements of the dish, and then remove the PBS from the plate using an aspirator.</li>
	<li>Add 1 mL of trypsin (10x dilution) to the dish. Incubate the plate until gentle tapping of the plate dislodges the cells. Take 10 mL of DMEM culture medium supplemented with 8% FBS and mechanically harvest the cells off the dish.</li>
	<li>Transfer the cell suspension into a 15 mL tube.</li>
	<li>Take 500 &#181;L of the cell suspension and put it in a cuvette for counting. Count the number of cells per mL of cell suspension. An automated cell counter was used for this study (see Table of Materials).</li>
	<li>Calculate the volume necessary to take from the cell suspension to have 1,00,000 cells.</li>
	<li>Take a 6-well plate and add the calculated volume of the cell suspension into each well, then add DMEM culture media supplemented with 8% FBS to 2 mL in each well.</li>
</ol>
2. Treatment of the cells
NOTE: The control and treatments are conducted in triplicate. The groups are as follows: Control (DMSO), 1 &#181;M of erastin, 0.3 &#181;M of RSL3, 250 &#181;M of FAC, 2 &#181;M of Ferrostatin 1, 1 &#181;M of erastin + 2 &#181;M of Ferrostatin 1, 0.3 of &#181;M RSL3 + 2 &#181;M of Ferrostatin 1, 250 &#181;M of FAC + 2 &#181;M of Ferrostatin 1. Four 6-well plates are needed for the experiment, as indicated in Table 1). The commercial details of all the necessary reagents are listed in the Table of Materials.
<ol>
	<li>24 h post-seeding, add the corresponding treatment to the well with the cells.</li>
	<li>Leave the dishes at 37 &#176;C and 5% CO2 in the incubator.</li>
</ol>
3. Lipid hydroperoxides staining of the treated cells with BODIPY 581/591 C11 probe
NOTE: The stock solution of the lipid hydroperoxide-specific probe is prepared in DMSO at a concentration of 1 mM. Aliquots of the stock solution are stored at -20 &#176;C in non-transparent tubes. For staining, prepare a 2 &#181;M working solution of the probe in DMEM media supplemented with 8% FBS.
<ol>
	<li>6 h post-treatment, observe the cells under a light microscope (cell rounding should be visible).</li>
	<li>Remove the dishes from the incubator and place them in a tissue culture laminar flow hood. 
	Using an aspirator, remove the media and floating (dead) cells.</li>
	<li>Gently add 2 mL of PBS to each well, wash the bottom with circular movements of the 6-well plates, and then remove the PBS from the wells using an aspirator.</li>
	<li>Add 2 mL of the working solution of the probe (2 &#181;M final concentration in DMEM supplemented with FBS).</li>
	<li>Leave the dish in the incubator at 37 &#176;C and 5% CO2 for 30 min in the dark (the plates can be wrapped in aluminum foil).</li>
</ol>
4. Flow cytometry analysis of lipid hydroperoxide content in the treated cells
NOTE: All the following steps are performed in the dark (no lights in the laminar flow hood).
<ol>
	<li>Take the dishes out of the incubator and place them in a tissue culture laminar flow hood. 
	Using an aspirator, remove the staining solution.</li>
	<li>Gently add 2 mL of PBS to each well, wash the bottom with circular movements of the 6-well plates, and then remove the PBS from the wells using an aspirator.</li>
	<li>Repeat the washing step one more time and aspirate any remaining PBS from the well using an aspirator.</li>
	<li>Add 150 &#181;L of a commercially available cell dissociation reagent (see Table of Materials) at the bottom of each well and incubate the plate until gentle tapping of the plate dislodges the cells. Take 250 &#181;L of FACS buffer (PBS, 2 mM EDTA, 0.5% BSA) and mechanically harvest the cells off the wells.</li>
	<li>Transfer the cells into the corresponding (previously marked) FACS tube with the filter cap. Place the tube with the cells on ice in the dark until the analysis is performed (analysis should be conducted within an hour after staining). 
	NOTE: FACS machine setup and calibration depend on the machine used (see Table of Materials).</li>
	<li>Create a new experiment and give it a name (Ferroptosis in DAOY - lipid hydroperoxides).</li>
	<li>In the experiment design, choose the laser (488 nm) and the filter (FITC).</li>
	<li>In view data, select the proper cell size (&#62;12 &#181;m), set the PMT voltages (the cell population should appear in the first quadrant of the SSC-H/FSC-H dot plot), and gate the dot plot.</li>
	<li>Add a new plot - histogram, with FITC-A on the y-axis and logarithmic scale.</li>
	<li>Vortex the samples in the FACS tube, place them in the FACS machine and load the sample. Record 10,000 FSC singlet events and name the file (e.g., DAOY WT CTL 6h). Export the file as .fcs.</li>
</ol>
5. Propidium iodide (PI) staining of the dead cells upon treatment
NOTE: The experiment design is exactly the same as for lipid hydroperoxide measurement (see step 1 and step 2).
<ol>
	<li>24 h post-seeding, take the dishes out of the incubator and place them in the laminar flow hood.</li>
	<li>Collect the media (with dead cells) in a 15 mL tube.</li>
	<li>Add 1 mL of PBS to each well, wash the bottom with circular movements of the 6-well plates, and then collect the PBS into the tube with the respective media.</li>
	<li>Add 200 &#181;L of trypsin (10x dilution) at the bottom of each well and incubate the plate until gentle tapping of the plate dislodges the cells. Use the respective media + PBS to harvest the cells off the wells.</li>
	<li>Centrifuge the cell suspension at 180 x g for 10 min at room temperature. Remove the supernatant using an aspirator.</li>
	<li>Resuspend the cell pellet in 300 &#181;L of FACS buffer and place it on ice until analysis.</li>
	<li>Just before the analysis, add PI solution (see Table of Materials) to a final concentration of 2 &#181;g/mL.</li>
</ol>
6. Flow cytometry analysis of dead cells 24h post-treatment
NOTE: FACS machine settings and calibration are done as previously indicated (see step 4).
<ol>
	<li>Create a new experiment and give it a name (Ferroptosis in DAOY - cell death).</li>
	<li>In the experiment design, choose the laser (488 nm) and the filter (PE).</li>
	<li>In view data, select the proper cell size (&#62;12 &#181;m), set the PMT voltages (the cell population should appear in the first quadrant of the SSC-H/FSC-H dot plot), and gate the dot plot.</li>
	<li>Add a new plot - histogram, with PE-A on the y-axis and logarithmic scale.</li>
	<li>Vortex the samples in the FACS tube, place them in the FACS machine and load the sample.</li>
	<li>Record 10,000 FSC singlet events and name the file (e.g., DAOY WT CTL 6h). Export the file as .fcs.</li>
</ol>
7. Flow cytometry analysis of lipid hydroperoxide content in the DAOY xCT-/- cells
NOTE: xCT-/- cells have been generated as previously described24.
<ol>
	<li>For this experiment, seed the cells as indicated in step 2, but instead of the treatment, supplement the media with 1 mM NAC overnight.</li>
	<li>The next day, maintain NAC in one group and remove it from the other.</li>
	<li>Perform the flow cytometry analysis of lipid hydroperoxide content in the exact same way as described in step 6.</li>
</ol>
8. Analysis of the flow cytometer results
<ol>
	<li>Open the .fcs document in the FlowJo software (see Table of Materials).</li>
	<li>Double click on the individual file to open all events (not only FCS singlets) recorded in the individual sample (SSC-A/FSC-A dot plot). Since the settings are such that the gating done on the machine is not preserved, it is necessary to make the gate once again and name the population (e.g., DAOY WT).</li>
	<li>Double click on the gated population to open another window with the histogram.</li>
	<li>Change the Y-axis to the fluorescent filter used (Comp-FITC/PE-A).</li>
	<li>Change the X-axis to modal (normalize each peak to its mode, i.e., to % of the maximum number of cells found in a particular bin).</li>
	<li>Copy the histogram into the layout editor. Repeat this for all the samples, and every time a new histogram is pasted in the layout editor, drag it over the previous one so that the histograms are overlaid (half-offset).</li>
</ol>

FACS Analysis of Lipid Hydroperoxides in Ferroptotic Medulloblastoma Cells

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We declare no conflict of interest for the study presented herewith.

The primary hallmark of ferroptotic cell death is the uncontrolled accumulation of lipid hydroperoxides in the plasma membrane. This oxidative damage may occur in an enzymatic or non-enzymatic manner, but in either case, the reaction is iron-dependent/catalyzed, which explains the name of this type of cell death. Lipid hydroperoxidation is often indirectly estimated by measuring the degradation products of lipid hydroperoxidation, such as 4-hydroxy-2,3-trans-nonenal (4-HNE) or malonaldehyde (MDA). These products, generat...

Ferroptosis is a newly contextualized, reactive oxygen species (ROS)-dependent type of cell death1. Besides ROS, iron plays a crucial role(s) in this type of cell death, hence the name2. The final and executive step of ferroptosis is the iron-catalyzed accumulation of oxidative damage of lipids in the plasma membrane that eventually leads to compromised membrane integrity and selective permeability, and, finally, cell death by bubbling. Lipid hydroperoxidation event is a naturally occurring phenomenon; however, its propagation throughout the cellular membrane is prevented by the antioxidant defense of the cell. The major player in this context is Se-protein glutathione peroxidase 4 (GPx4), which can approach the membrane and convert lipid hydroperoxides into their less toxic alcohol derivatives3. The reducing power for GPx4 is mainly, but not exclusively, provided by glutathione (GSH), a tripeptide composed of the non-essential amino acids: glycine, glutamate, and cysteine. The rate-limiting amino acid for the biosynthesis of GSH is cysteine4. Although cysteine is classified as a non-essential amino acid, its requirements can easily exceed its internal production in highly proliferative cells (such as cancer cells). It thus has been re-classified in the group of semi-essential amino acids. The necessary import of cysteine occurs mainly through the Xc- system, which allows the import of the oxidized (dominant) form of cysteine (aka cystine) at the expense of glutamate export5. The Xc- system is composed of a Na+-independent, Cl--dependent transport subunit, known as xCT, and a chaperon subunit, known as CD98. Until recently, the anti-ferroptotic properties of the xCT-GSH-GPx4 axis have been seen as unique and irreplicable6. However, in 2019, an alternative anti-ferroptotic pathway, consisting of ubiquinol (Coenzyme Q10) and its regenerative enzyme - ferroptosis suppressor protein 1 (FSP1), has been described7,8. Soon afterward, yet another lipid hydroperoxide detoxifying system involving GTP cyclohydrolase-1/tetrahydrobiopterin (GCH1/BH4) has been reported9. Nonetheless, the central role of the xCT-GSH-GPx4 axis in the prevention of ferroptosis seems not to be challenged.
Over the past decade, ferroptosis has been extensively studied in a variety of tumor types, showing great potential as an anti-cancer strategy (reviewed by Lei et al.10). Furthermore, it has been reported that cancer cells exhibiting high resistance to conventional chemotherapeutics and/or a propensity to metastasize are surprisingly sensitive to ferroptosis inducers, such as inhibitors of GPx411,12,13. However, in the context of brain tumors, the potential of ferroptotic inducers remains largely understudied. While this type of cell death has been closely associated with cerebral ischemia-reperfusion injury14and neurodegenerative diseases15, its potential in the context of brain tumors has mainly been limited to glioblastoma, the most common malignant craniocerebral tumor (reviewed by Zhuo et al.16). On the other hand, the sensitivity of medulloblastoma, the most common malignant pediatric brain tumor and a leading cause of childhood mortality, to ferroptosis inducers remains largely unexplored. To the best of our knowledge, there is scarce peer-reviewed literature linking ferroptosis and medulloblastoma. Nonetheless, some studies have revealed that iron plays a crucial role in the survival, proliferation, and tumorigenic potential of both medulloblastoma and glioblastoma cancer stem cells (CSCs)17,18, potentially rendering them more vulnerable to ferroptosis induction. This is particularly significant as medulloblastoma is notorious for its subpopulation of CSCs, or tumor-initiating/propagating cells, which appear to be largely responsible for tumor chemoresistance, dissemination, and relapse19.
Sensitivity to ferroptosis induction is typically investigated by measuring lipid hydroperoxide content/accumulation, which may or may not lead to cell death. The most commonly used ferroptosis inducers are (1) erastin, an inhibitor of the xCT transporter20,(2) RSL3, an inhibitor of the GPx4 enzyme2, and/or (3) iron donors, such as ferro-ammonium citrate (FAC)21. Lipid hydroperoxide content is assessed using the selective probe BODIPY 581/591 C1122, which has excitation and emission maxima at 581/591 nm in its reduced state. Upon interaction with and oxidation by lipid hydroperoxides, the probe shifts its excitation and emission maxima to 488/510 nm. Typically, a significant increase in lipid hydroperoxide content precedes ferroptotic cell death. Since ferroptosis is not a programmed cell death, there is no molecular signaling cascade leading to its execution. Therefore, the only way to confirm it is to monitor lipid hydroperoxide content and use specific inhibitors for this type of cell death, such as ferrostatin 123. Ferrostatin 1 is a lipophilic antioxidant that can penetrate the lipid compartment of the cell and detoxify lipid hydroperoxides, thereby preventing ferroptotic events.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BODIPY 581/591 C11</td>
			<td>Thermo Fisher</td>
			<td>D3861</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Beckman</td>
			<td>Coulter Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>Erastin</td>
			<td>Sigma-Aldrich</td>
			<td>E7781-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferroamminium citrate</td>
			<td>Acros Organics</td>
			<td>211842500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrostatin-1</td>
			<td>Sigma-Aldrich</td>
			<td>SML0583-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovin serum (FBS)</td>
			<td>Dominique Dutcher</td>
			<td>500105N1N</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>FACS Melody</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermo Fisher</td>
			<td>11599686</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A7250</td>
			<td></td>
		</tr>
		<tr>
			<td>PlasmoTest Mycoplasma Detection Kit</td>
			<td>InvivoGen</td>
			<td>rep-pt1</td>
			<td></td>
		</tr>
		<tr>
			<td>propidium iodide</td>
			<td>Invitrogen</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>RSL3</td>
			<td>Sigma-Aldrich</td>
			<td>SML2234-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin - EDTA 10X - 100 mL</td>
			<td>Dominique Dutcher</td>
			<td>X0930-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

revealing the ferroptotic phenotype of medulloblastoma

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named &#34;ferroptosis.&#34; The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic &#34;bubbling&#34; phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

The medulloblastoma cell line DAOY was cultured in a standard DMEM medium supplemented with 8% FBS until it reached approximately 60% confluency. On the day of the experiment, cells were harvested, and 1,00,000 cells per well were plated in 6-well plates, according to Table 1. The following day, cells (in triplicate) were treated with either 1 &#181;M of erastin, 0.3 &#181;M of RSL3, or 250 &#181;M of FAC. The plates were then placed in the incubator at 37 &#176;C and 5% CO2. After 6 h, cells ...

Read this Scientific Article Protocol about Author Spotlight: Tracing the Ferroptotic Signatures and Cell Death Dynamics in Medulloblastoma for Advanced Therapeutics at JoVE.com

Author Spotlight: Tracing the Ferroptotic Signatures and Cell Death Dynamics in Medulloblastoma for Advanced Therapeutics 

Lipid hydroperoxide content represents the most commonly used indicator of ferroptotic cell death. This article demonstrates the step-by-step flow cytometry analysis of lipid hydroperoxide content in cells upon ferroptosis induction.

Revealing the Ferroptotic Phenotype of Medulloblastoma

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and ...

revealing-the-ferroptotic-phenotype-of-medulloblastoma

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Cancer Research

780 Views.  Centre Scientifique de Monaco (CSM). Lipid hydroperoxide content represents the most commonly used indicator of ferroptotic cell death. This article demonstrates the step-by-step flow cytometry analysis of lipid hydroperoxide content in cells upon ferroptosis induction.

Video: Author Spotlight: Tracing the Ferroptotic Signatures and Cell Death Dynamics in Medulloblastoma for Advanced Therapeutics 

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells (CSCs) and identifying suitable molecular targets relies on methods that allow their specific discrimination in heterogeneous samples such as cell lines and ex vivo tumor tissue. Flow cytometry/FACS is a powerful technology to multi-parametrically dissect biological samples at the single cell level and is to date the method of choice to recover live cells for downstream analyses. Surface markers such as CD44 and CD133 as well as detection of aldehyde dehydrogenase enzymatic activity have often been used to define and sort out CSCs from tumor samples by FACS. A complementary approach, depicted here in methodological detail, makes use of functional dye extrusion by ABC drug transporters, which identifies a distinct population of fluorescence-dim cells commonly referred to as side population (SP). SP&#160;cancer cells exhibit canonical stem cell characteristics and can be abrogated and functionally confirmed using agents that inhibit the dye-extruding drug transporter (most frequently ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD338). Moreover, the SP&#160;assay is compatible with other flow cytometric evaluations such as staining of surface antigens, aldehyde dehydrogenase detection and dead cell discrimination (e.g., with 7-AAD or propidium iodide (PI)). Thus, we describe a valuable and broadly applicable method for CSC identification, isolation and sub-characterization mechanistically based on a functional, rather than a phenotypic parameter. Although originally performed with Hoechst 33342 as triggering dye, we here focus on the more recent Violet dye-based SP phenotype that is resolvable on any flow cytometer equipped with a violet laser source.

Methods allowing the characterization and isolation of stem cell populations from biological samples are critical for the advance of stem cell-targeted treatments in cancer and beyond. Here, we provide a detailed protocol for cancer stem cell isolation using the dye-triggered side population phenotype.

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

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 Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...