This study is fully supported by the Division of Intramural Research of the National Institute of Nursing Research of the NIH, Bethesda, Maryland.

The current study (NCT00852111) was approved by the Institutional Review Board (IRB) of the National Institutes of Health (NIH), Bethesda, Maryland. Participants enrolled in this study were euthymic men, 18 years of age or older, who were diagnosed with non-metastatic prostate cancer with or without prior prostatectomy and were scheduled to receive external beam radiation therapy (EBRT). Potential participants were excluded if they had a progressive disease that could cause significant fatigue, had psychiatric disease within the past five years, had uncorrected hypothyroidism or anemia, or had a second malignancy. Individuals who used sedatives, steroids, or non-steroidal anti-inflammatory agents were also excluded. Healthy control blood samples were obtained at the NIH Department of Transfusion medicine from healthy donors under an IRB-approved protocol (NCT00001846). All participants are recruited at the Magnuson Clinical Research Center at the NIH. Signed written informed consents were obtained prior to study participation.
1. Mitochondrial Function Measurement Preparation (Day 1 of the Experiment)
<ol>
	<li>Hydrate Sensor Cartridge (see Table of Materials; approximate duration: 5 min).
	<ol>
		<li>Remove Sensor Cartridge from package. Add 200 &#956;L Calibrant solution into each well of the utility plate, then fill each moat with 400 &#956;L Calibrant solution.</li>
		<li>Return Sensor plate to the utility plate, which now has Calibrant solution in it. Hydrate the Cartridge in a non-CO2 37 &#176;C incubator overnight. 
		NOTE: Sensor Cartridge needs to be hydrated for a minimum of 4 h and a maximum of 72 h.</li>
	</ol>
	</li>
</ol>
2. Clinical Sample Preparation (Day 2 of the Experiment)
<ol>
	<li>Measure fatigue using the 13-item Functional Assessment of Chronic Illness Therapy - Fatigue (FACIT-F)2,23,24 at baseline (prior to EBRT initiation), midpoint and completion of EBRT, and 1-year post-EBRT.
	<ol>
		<li>Use a 0 - 4 scale for each item response, where a 0 represents &#34;not at all&#34; and a 4 indicates that the respondent relates to the corresponding statement &#34;very much.&#34; Total scores should range from 16 - 53, with lower scores reflecting high fatigue intensity.</li>
		<li>Define fatigue as a FACIT-F score lower than 43, with a FACIT-F score of &#8805;43 indicating absence of or not clinically-meaningful fatigue2. 
		NOTE: A FACIT-F score of 43 best divides fatigue scores of cancer patients and the general population2.</li>
		<li>Include the following 13 items in the FACIT Fatigue Scale: 1) I feel fatigued; 2) I feel weak all over; 3) I feel listless (&#34;washed out&#34;); 4) I feel tired; 5) I have trouble starting things because I&#39;m tired; 6) I have trouble finishing things because I am tired; 7) I have energy; 8) I am able to do my usual activities; 9) I need to sleep during the day; 10) I am too tired to eat; 11) I need help doing my usual activities; 12) I am frustrated by being too tired to do the things I want to do; and 13) I have to limit my social activity because I am tired.</li>
	</ol>
	</li>
	<li>Isolate PBMC from freshly collected blood samples (approximate duration: 1 h).
	<ol>
		<li>Collect 8 mL of blood into a Mononuclear Cells Preparation Tube (see Table of Materials). 
		NOTE: Blood samples should be processed within 2 h of collection. The quality of PBMCs may be compromised if blood samples are processed more than 2 h after sample collection.</li>
		<li>Centrifuge at 1,750 x g for 30 min at room temperature (18 - 25 &#176;C). Transfer the cloudy layer to a 15 mL conical tube. Add up to 15 mL of PBS and invert 5 times.</li>
		<li>Centrifuge at 300 x g for 15 minutes at 4 &#176;C. Carefully remove and discard the supernatant without disturbing pellet. Re-suspend the pellet by adding up to 10 mL PBS, and invert 5 times.</li>
		<li>Centrifuge at 300 x g for 10 min at 4 &#176;C. Discard liquid supernatant and re-suspend cells in 1 mL PBS. Transfer the PBMCs to a 1.5 mL microfuge tube.</li>
		<li>Centrifuge at 610 x g for 10 minutes. Carefully remove supernatant and resuspend pellet in complete RPMI-1640 (RPMI-1640 supplemented with 10% FBS, 10 mM Penicillin/Streptomycin).</li>
	</ol>
	</li>
	<li>Coat cell plates for non-adherent cells (approximate duration: 30 min).
	<ol>
		<li>Prepare cell and tissue adhesive solution (see Table of Materials) by diluting the stock solution in 0.1 M sodium bicarbonate pH 8.0. The working solution should be at 3.5 &#956;g/cm2 of surface area. 
		NOTE: This solution contains polyphenolic proteins extracted from the marine mussel, Mytilus edulis. These proteins are key components of the glue secreted by the mussel to anchor itself to surfaces. We find that Cell-Tak works the best with PBMCs.</li>
		<li>Add 100 &#956;L of the diluted adhesive solution to each well and incubate for at least 20 min at room temperature. Wash three times with DI water and air dry.</li>
	</ol>
	</li>
</ol>
3. Mitochondrial Function Measurement
<ol>
	<li>Preparation of Assay Media (approximate duration: 10 min). 
	NOTE: Assay media must be freshly prepared on the day of the experiment.
	<ol>
		<li>Add L-glutamine, pyruvate, and glucose to base media (the same constituents as Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), but without any sodium bicarbonate, glucose, glutamine, or sodium pyruvate) to make assay media. Warm media up to 37 &#176;C, then adjust pH to 7.4. 
		NOTE: Concentrations of L-glutamine, pyruvate, and glucose are usually the same as concentrations in normal growth media, but can be adjusted based on the assay.</li>
	</ol>
	</li>
	<li>Prepare PBMCs for the Mito Stress test (approximate duration: 2 h).
	<ol>
		<li>Plate enough PBMCs from Step 2 into each well to reach 80 - 90% confluency. In our experience, 1.5 x 105 cells/well yielded the most consistent results. 
		NOTE: Wells A and H are background wells and should only contain assay media with no cells. In wells B to G, we recommend plating at least 3 - 6 wells per patient sample in order to account for outliers.</li>
		<li>Spin plates down at 200 x g for 2 min to allow cells to adhere to the bottom of the wells. Wash the cells once with Assay media. This step removes serum and sodium bicarbonate from the growth media. 
		NOTE: In our experience, the washing step typically removes 20 - 30% of cells.</li>
		<li>Add 180 &#956;L Assay media into each well, including background wells A and H. Incubate in a non-CO2 37 &#176;C incubator for 45 - 60 min.</li>
	</ol>
	</li>
	<li>Running the Mito Stress Test
	<ol>
		<li>Reconstitute drugs in the Mito Stress Kit with assay media as follows:
		<ol>
			<li>Oligomycin: Add 252 &#956;L assay media into the vial to generate a stock solution at 50 &#956;M.</li>
			<li>FCCP: Add 288 &#956;L of assay media into the vial to generate a stock solution at 50 &#956;M.</li>
			<li>Antimycin A/Rotenone: Add 216 &#956;L of assay media into the vial to generate a stock solution at 25 &#956;M.</li>
		</ol>
		</li>
		<li>Vortex reconstituted drugs for approximately 1 minute. Dilute into working solutions. 
		NOTE: Concentrations of the working solutions should be titrated and pre-determined prior to the experiment depending on the cell type. For PBMCs, we find that 1 &#956;M of Oligomycin, 1 &#956;M FCCP, and 0.5 &#956;M antimycin A/Rotenone work the best.</li>
		<li>Pipette the drugs into each port in the sensor cartridge sequentially:
		<ol>
			<li>Port A: Pipette 20 &#956;L of 10 &#956;M Oligomycin, for a final concentration in each well of 1 &#956;M.</li>
			<li>Port B: Pipette 22 &#956;L of 10 &#956;M FCCP, for a final concentration in each well of 1 &#956;M.</li>
			<li>Port C: Pipette 25 &#956;L of 5 &#956;M antimycin A/rotenone for a final concentration in each well of 0.5 &#956;M.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Select &#34;Mito Stress Test&#34; on an extracellular flux instrument. Follow instrument prompt and insert the sensor cartridge. The instrument will automatically perform sensor calibration. Insert cell plate after sensor calibration, and the instrument will finish the rest of the assay. Oxygen dynamics is measured at 530 nm (excitation)/650nm (emission), and proton concentration is measured at 470 nm (excitation)/ 530 nm (emission).</li>
</ol>
4. Normalization of Mitochondrial Function Data
<ol>
	<li>Prepare Cell Proliferation Assay solution (approximate duration: 5 min).
	<ol>
		<li>Add 48 &#956;L nucleic acid stain (500x) and 240 &#956;L background suppressor to 11.7 mL PBS (2x). The nucleic acid stain is a cell-permeant DNA-binding dye, and the background suppressor is a masking dye which blocks dead cells or cells with compromised cell membrane integrity from being stained. The combination of the DNA-binding dye and the background suppressor ensures that only live cells are stained.</li>
		<li>Add equal volume of 2x&#160;working solution (180 &#956;L) directly into the medium in each well after the Mito Stress Test has completed.</li>
	</ol>
	</li>
	<li>Incubate the cells in the presence of cell proliferation assay solution in a 37 &#176;C incubator for 45 - 60 min. Quantify the number of live cells (fluorescent) on a plate reader at 508 nm (excitation)/527 nm (emission) and normalize OCR data (approximate duration: 10 min). 
	NOTE: in addition to the number live cells, users may also choose to normalize their data with the total number of cells, amount of nucleic acid, protein concentrations, etc.</li>
</ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Predictors+of+Fatigue+in+Men+With+Non-Metastatic+Prostate+Cancer+Receiving+External+Beam+Radiation+Therapy.">Clinical Predictors of Fatigue in Men With Non-Metastatic Prostate Cancer Receiving External Beam Radiation Therapy.</a> Clinical Journal of Oncology Nursing. 19 (6), 744-750 (2015).</li><li>Feng, L. R., Wolff, B. S., Lukkahatai, N., Espina, A., Saligan, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploratory+Investigation+of+Early+Biomarkers+for+Chronic+Fatigue+in+Prostate+Cancer+Patients+Following+Radiation+Therapy.">Exploratory Investigation of Early Biomarkers for Chronic Fatigue in Prostate Cancer Patients Following Radiation Therapy.</a> Cancer Nursing. 40 (3), 184-193 (2017).</li><li>Myhill, S., Booth, N. 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The authors have nothing to disclose.

Fatigue in cancer patients is a debilitating condition that is not well defined or characterized1. Diagnosis of fatigue entirely relies on subjective reporting and there is no current diagnostic standard or treatment for this condition, largely due to a lack of understanding in its pathobiology2. Of the proposed mechanisms underlying fatigue in cancer patients, impairment in mitochondrial function is one of the most therapeutically targetable pathways. Therefore, we develop...

Fatigue is a prevalent and distressing condition that has a negative impact on the quality of life of cancer patients1. To this date, cancer fatigue remains poorly defined and relies only on subjective reporting by patients2. Therefore, there is an urgent need to identify an easily adaptable diagnostic laboratory test to objectively characterize fatigue in the clinical setting3,4.
Multiple underlying mechanisms, including mitochondria dysfunction, have been proposed to cause fatigue5. Mitochondria are the powerhouse organelles, providing 95% of cellular energy needs via oxidative phosphorylation, and play an important role in calcium signaling, apoptosis, immune signaling, and regulation of other intracellular signaling events6. Accordingly, impaired mitochondrial bioenergetics and defects in energy production may contribute to fatigue. Supporting this hypothesis, previous studies have observed mutations in mitochondrial DNA in patients with chronic fatigue syndrome7. While it remains unclear whether the pathophysiological origin of fatigue lies within the central nervous system or peripheral tissues, such as skeletal muscles8,9, there is currently no direct method to accurately assess mitochondrial dysfunction related to fatigue in live, respiring cells.
Using peripheral blood mononuclear cells (PBMCs) to study mitochondrial function offers several advantages. First, PBMCs are readily available in the clinical setting using standard phlebotomy and can be isolated quickly using basic laboratory techniques. Second, blood collection is less invasive than collecting other tissues such as a muscle biopsy. Thus, blood samples can be collected from the same patient repeatedly over time, which facilitates longitudinal assessment of treatment effects. Interestingly, mitochondrial function in PBMCs appeared to be well correlated with kidney mitochondrial status in an animal model10. Furthermore, immune cell mitochondria have been used as a proxy for detecting systemic changes under different disease conditions11,12. Mitochondria in circulating immune cells are particularly sensitive to changes in immune functions and immune signaling molecules such as cytokines13,14,15. For example, it has been observed that PBMCs from patients with acute rheumatic inflammatory diseases exhibit high baseline oxygen consumption14. In contrast, oxygen consumption was reduced in PBMCs isolated from patients with systemic inflammatory conditions including sepsis16. Under inflammatory conditions, free radicals produced by dysfunctional mitochondria may further contribute to elevated oxidative stress and prolonged inflammation17. The central role of mitochondria in energy production as well as in oxidative stress suggests the potential utility of using mitochondrial function as a proxy for studying fatigue in cancer patients 13.
Previous studies examining mitochondrial function utilized biochemical techniques, mitochondrial membrane potential measurement, or isolation of specific cell populations that may not be readily adaptable in the clinical setting5,14,18. In recent years, the development of extracellular flux assays has allowed researchers to easily and accurately examine changes in oxygen consumption rate (OCR) in response to automated injections of respiratory inhibitors19,20,21,22. However, most of these studies are designed for specific cell types and the large high-throughput format may not be applicable in a clinical setting. In this manuscript, we describe an optimized protocol for examining mitochondrial function for clinical use.

<table border="0" cellpadding="0" cellspacing="0" style="width:876px;" width="875">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:331px;">CPT Mononuclear Cells Preparation Tube&#160;</td>
			<td style="width:123px;">BD Biosciences</td>
			<td style="width:165px;">362761</td>
			<td style="width:257px;">For isolating PBMCs following phlebotomy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RPMI-1640&#160;</td>
			<td>Corning</td>
			<td>10-040</td>
			<td>For making growth media for PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td>For making growth media for PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>For making growth media for PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell-Tak</td>
			<td>Corning</td>
			<td>354240</td>
			<td>Cell and Tissue adhesive solution; allows suspension cells to adhere to the surface</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Seahorse XF Calibrant Solution</td>
			<td>Agilent</td>
			<td>103059-000</td>
			<td>For hydrating cartridges</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XFp Fluxpak (miniplates and sensor cartridges)</td>
			<td>Agilent</td>
			<td>103022-100</td>
			<td>Contains XFp cell culture miniplates and sensor cartridges</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XF base media</td>
			<td>Agilent</td>
			<td>103335-100</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">45% cell culture D-(+)-Glucose solution</td>
			<td>Corning</td>
			<td>25-037-CI</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium pyruvate solution</td>
			<td>Corning&#160;</td>
			<td>25-000-CI</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-glutamine solution</td>
			<td>ThermoFisher</td>
			<td>25030081</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Seahorse XFp Mito Stress Test Kit</td>
			<td>Agilent</td>
			<td>103010-100</td>
			<td>Contains oligomycin, FCCP, antimycin A/rotenone</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CyQUANT Direct Cell Proliferation Assay</td>
			<td>ThermoFisher</td>
			<td>C35011</td>
			<td>For quantification of live cells and data normalization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Seahorse XFp Analyzer</td>
			<td>Agilent</td>
			<td>S7802AEA</td>
			<td>For measuring mitochondrial function in live cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cytation 5 Cell Imaging Multi-Mode Reader (or any instrument that can quantify fluorescent cells in a plate)</td>
			<td>BioTek</td>
			<td>BTCYT5PV</td>
			<td>For quantification of live cells and data normalization</td>
		</tr>
	</tbody>
</table>

evaluating the role of mitochondrial function in cancer-related fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

The Mito Stress Test relies on measuring oxygen consumption rate (OCR) after sequential injection of various respiratory inhibitors to map a complete mitochondrial profile. OCR measurements after each drug injection can be used to calculate the following parameters related to mitochondrial health: Basal OCR is first measured before any drug injection to assess oxygen consumption needed to meet resting level ATP demand. Basal respiration is calculated by subtracting non-mi...

Watch this Scientific Journal Video about Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue at JoVE.com

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

evaluating-role-mitochondrial-function-cancer-related

Research

JoVE Journal

Cancer Research

9.1K Views.  National Institutes of Health. Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Video: Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Integration of Bioinformatics Approaches and Experimental Validations to Understand the Role of Notch Signaling in Ovarian Cancer

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to interference with proper development and may even result in initiation or progression of cancers in certain cases. Because this pathway serves complex and versatile functions, it can be studied extensively through many different approaches. Of these, bioinformatics provides an undeniably cost-efficient, approachable, and user-friendly method of study. Bioinformatics is a useful way to extract smaller pieces of information from large-scale datasets. Through the implementation of various bioinformatics approaches, researchers can quickly, reliably, and efficiently interpret these large datasets, yielding insightful applications and scientific discoveries. Here, a protocol is presented for integration of bioinformatics approaches to investigate the role of Notch signaling in ovarian cancer. Furthermore, bioinformatics findings are validated through experimentation.

Bioinformatics is a useful way to process large-scale datasets. Through the implementation of bioinformatics approaches, researchers can quickly, reliably, and efficiently obtain insightful applications and scientific discoveries. This article demonstrates the utilization of bioinformatics in ovarian cancer research. It also successfully validates bioinformatics findings through experimentation.

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to ...

Measuring the Motor Aspect of Cancer-Related Fatigue using a Handheld Dynamometer

Cancer-related fatigue (CRF) is commonly reported by patients both during and after receiving treatment for cancer. Current CRF diagnoses rely on self-report questionnaires which are subject to report and recall biases. Objective measurements using a handheld dynamometer, or handgrip device, have been shown in recent studies to correlate significantly with subjective self-reported fatigue scores. However, variations of both the handgrip fatigue test and fatigue index calculations exist in the literature. The lack of standardized methods limits the utilization of the handgrip fatigue test in the clinical and research settings. In this study, we provide detailed methods for administering the physical fatigue test and calculating the fatigue index. These methods should supplement existing self-reported fatigue questionnaires and help clinicians assess fatigue symptom severity in an objective and quantitative manner.

Simple and accessible methods were developed to measure the motor aspect of cancer-related fatigue objectively and quantitatively. We describe, in detail, ways to administer the physical fatigue test using a simple handgrip device as well as methods to calculate fatigue indices.

Cancer-related fatigue (CRF) is commonly reported by patients both during and after receiving treatment for cancer. Current CRF diagnoses rely on ...

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 ...