This manuscript was supported in part by the Korea Medical Device Development Fund (KMDF, Grant No. RS-2020-KD000019) and the Korea Health Industry Development Institute (KHIDI, Grant No. HI19C0521020020).

All whole blood samples were obtained from lung cancer patients. The research and analysis at Clinomics are carried out by the Cancer Genomics Research Institute, and IRB research approval by the government is led by the Asan Medical Center Institutional Review Committee (IRB NO. 2021-0802) with the IRB number registered for research at Clinomics.
1. Sample preparation
<ol>
	<li>Collect 9 mL of whole blood into an EDTA or cfDNA-stable blood collection tube.</li>
	<li>Mix well by flipping the tube up and down approximately 10 times.</li>
	<li>Store the samples at room temperature (RT; for short-term storage) or 4 &#176;C (for long-term storage). Do not freeze and thaw.</li>
</ol>
2. Device preparation
<ol>
	<li>Press the power switch to turn on the instrument. 
	NOTE: A loading screen appears on the touchpad, and the instrument is initialized. Keep hands away from the instrument during the initialization. The cartridge selection screen appears after the instrument initialization is completed.</li>
	<li>Select the sample mode to be used. Change the number of samples by pressing the arrow.</li>
</ol>
3. Device operation and sample collection
<ol>
	<li>Loading of the LBx-1 cartridge
	<ol>
		<li>Select the cartridge type on the touchscreen panel of the instrument. Change the number of samples by pressing the arrow button.</li>
		<li>Open the door of the instrument and insert all the cartridges to be used in order of the number on the cartridge holder. Make sure to correctly insert the cartridge and the cartridge holder. If the cartridge is not inserted correctly, it may cause considerable damage to the instrument.</li>
		<li>For a total of four cartridges, place the dummy cartridge in the empty space of the cartridge holder.</li>
		<li>Use the support wheel to mount the cartridge and tighten the lock nut to secure it.</li>
		<li>Close the door and press the RUN button on the touchpad screen. The instrument closes the valves on the cartridges, which takes approximately 30 s.</li>
		<li>Follow the message to open the door, remove the support wheel, and remove the valve-closed cartridge from the cartridge holder.</li>
		<li>Place the cartridge on the table and prepare to inject the whole blood sample. Pipette a maximum of 10 mL of whole blood sample using a serological pipette. 
		CAUTION: Dangers of handling blood. During the entire procedure of handling blood samples and reagents, it is important to wear a laboratory coat and laboratory gloves. The provided MSDS should be thoroughly studied by the laboratory workers.</li>
		<li>Insert the pipette tip deep into the sample inlet of the cartridge and slowly inject the whole blood sample. 
		NOTE When using more than or equal to 9 mL of whole blood sample, no phosphate buffered saline (PBS) addition is necessary. When using less than 9 mL of whole blood sample, add PBS to make the total volume to 9 mL. For example, when using 7.2 mL of whole blood, please add 1.8 mL of PBS into the sample inlet. A serological pipette or a pipette tip can be used for the injection of whole blood and PBS. When using less than 8 mL of whole blood sample, the buffy coat may not be recovered even by adding 1 mL of PBS. For gDNA prep, use 200 &#181;L of whole blood, which was separated before this step.</li>
		<li>Insert the cartridges to be used in order of the number on the cartridge holder. Use the support wheel to mount the cartridge and tighten the lock nut to secure it. Close the door and press the OK button. 
		NOTE: Plasma and buffy coat are separated from whole blood automatically, which takes approximately 30 min. 
		CAUTION Danger during operation. Opening the door or touching the instrument during high-speed rotating operations can cause serious injuries. There is also a risk of injury due to asymmetric loading of the rotor. If unusual vibrations and noises occur when the instrument starts at assisted cell enrichment or expert mode, cartridge placement may be asymmetrical. Immediately press the power key to stop and properly install the cartridge.</li>
		<li>Look for the message on the screen accompanied by an alarm sound, which appears when the separation of plasma and buffy coat is complete.</li>
		<li>Stop the alarm by opening the door or pressing the STOP button. Open the door, remove the cartridge, and place it on the table.</li>
		<li>Recover 3 mL of plasma from the plasma outlet using a 1 mL pipette tip. Recover the buffy coat of 3 mL from the buffy coat outlet using a 1 mL pipette tip.</li>
	</ol>
	</li>
	<li>Loading the LBx-2 cartridge
	<ol>
		<li>Select the cartridge name on the touchscreen panel of the instrument. Change the number of samples by pressing the arrow button.</li>
		<li>Open the door and insert the cartridges to be used in order of the number on the cartridge holder.</li>
		<li>For a total of four cartridges, place the dummy cartridge in the empty space of the cartridge holder.</li>
		<li>Use the support wheel to mount the cartridge and tighten the lock nut to secure it. Ensure to correctly insert the cartridge in the cartridge holder. If the cartridge is not inserted correctly, it may cause considerable damage to the instrument.</li>
		<li>Close the door and press the RUN button on the touchpad screen. The instrument closes the valves on the cartridge, which takes approximately 30 s.</li>
		<li>Follow the message to open the door, remove the support wheel, and remove the valve-closed cartridge from the cartridge holder and place it on the table.</li>
		<li>Place the cartridge on the table and prepare to inject the density gradient solution and the whole blood sample. Check the volume of the density gradient solution and PBS to be injected depending on the volume of the whole blood sample (Supplementary Table 1).</li>
		<li>Pipette the density gradient solution using a serological pipette. Insert the pipette tip deep into the inlet of the cartridge and slowly inject the density gradient solution.</li>
		<li>After injecting the density gradient solution, pipette the whole blood sample using a serological pipette. Insert the pipette tip deep into the sample inlet of the cartridge and slowly inject the whole blood sample. 
		NOTE: When using less than 9 mL of the whole blood sample, add PBS by referring to this table (Supplementary Table 1).</li>
		<li>Insert the cartridges to be used in order according to the number on the cartridge holder. Use the support wheel to mount the cartridge and tighten the lock nut to secure it. Close the door and press the OK button.</li>
		<li>Plasma and PBMC are separated from whole blood automatically, which takes approximately 30 min. Look for the message that appears accompanied by an alarm sound, when the separation of plasma and PBMC is complete.</li>
		<li>Stop the alarm by opening the door or pressing the STOP button. Open the door, remove the cartridge, and place it on the table.</li>
		<li>Recover 3 mL of plasma from the plasma outlet using a 1 mL pipette tip. Recover 3 mL of the PBMC from the PBMC outlet using a 1 mL pipette tip.</li>
	</ol>
	</li>
	<li>Loading the FAST-auto cartridge
	<ol>
		<li>Select the cartridge on the touch screen panel of the instrument. Change the number of samples by pressing the arrow button.</li>
		<li>Open the door and insert the cartridges to be used in order of the number on the cartridge holder. For a total of four cartridges, place the dummy cartridge in the empty space of the cartridge holder.</li>
		<li>Use the support wheel to mount the cartridge and tighten the lock nut to secure it.</li>
		<li>Close the door and press the RUN button on the touch pad screen. The instrument closes the valves on the cartridge, which takes approximately 30 s.</li>
		<li>Follow the message to open the door, remove the support wheel, and remove the valve-closed cartridge from the cartridge holder.</li>
		<li>Place the cartridge on the table and prepare to inject the PBS solution and whole blood sample. Pipette 6 mL of PBS solution using a serological pipette. Insert the pipette tip deep into the PBS inlet of the cartridge and slowly inject 6 mL of the PBS solution.</li>
		<li>After injecting the PBS solution, pipette 3 mL of whole blood or PBMC sample obtained from LBx-2 using a serological pipette, which was previously rinsed with 1% BSA to prevent sticking of the residue. Insert the pipette tip deep into the sample inlet of the cartridge and slowly inject the whole blood sample.</li>
		<li>Insert the cartridges to be used in order of the number on the cartridge holder. Use the support wheel to mount the cartridge and tighten the lock nut to secure it. Close the door and press the OK button.</li>
		<li>CTCs are enriched from whole blood automatically, which takes approximately 15 min. Look for a message that appears accompanied by an alarm sound when the enrichment of CTCs is complete. Stop the alarm by opening the door or pressing the STOP button.</li>
		<li>Open the door, remove the cartridge, and place it on the table. Insert the back plate remover (BPR) into the four holes on the front of the cartridge. Press the dark blue wing with the thumbs of both hands, and then press the light blue body until it clicks.</li>
		<li>Carefully remove the cartridge body by lifting it up. Very gently pick up the filter membrane by the edge using a tweezer. Please make sure to use the outer rim (the 1 mm wide edge part) of the filter membrane to pinch and hold the filter membrane.</li>
		<li>Carefully place the filter membrane (on which enriched CTCs are residing) into a 1.5 mL tube for nucleic acid preparation. If necessary, recover the filtered blood to the blood outlet using a 1 mL pipette tip.</li>
	</ol>
	</li>
</ol>
4. Maintenance of the system
<ol>
	<li>Preparation for cleaning and disinfection of the instrument
	<ol>
		<li>Clean all the accessible surfaces of the instrument and the accessories once a week using ethanol and dried tissue, and also immediately when contaminated.</li>
		<li>Clean the bowl and rotor shaft regularly using alcohol (ethanol and isopropanol) or alcohol-based disinfectants.</li>
	</ol>
	</li>
	<li>Cleaning and disinfecting the instrument 
	NOTE: For general troubleshooting and other notes on the machine, refer to Supplementary Table 2.
	<ol>
		<li>Switch off the instrument using the main power switch. Disconnect the power plug from the power supply.</li>
		<li>Open the door and clean and disinfect all the accessible surfaces of the instrument, including the power cable, using a damp cloth and the recommended cleaning agents.</li>
		<li>Check the rotor shaft for damage. Inspect the instrument for corrosion and damage.</li>
		<li>Connect the instrument to the power supply only if it is fully dry inside and out.</li>
	</ol>
	</li>
	<li>Disconnect the main power plug and remove the fuse holder. The fuse holder is located above the power socket. Replace the used fuse with a spare one from the container.</li>
</ol>

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The authors have no conflicts of interest related to this work.

The amount and concentration of cfDNA and CTC depends on the individual, stage, and type of cancer. It also depends on the condition of the patient2,4,5,10,20. In particular, in the early or precancerous stages of cancer, the concentrations of cancer-related substances are very low, so there is a high possibility that it cannot be detected. Nevertheless, early...

Early and accurate detection of various diseases, including cancer, is the most important factor in establishing a treatment strategy1,2,3,4. In particular, early detection of cancer is closely related to increased survival chances for the patient5,6,7,8. Recently, liquid biopsies have been in the spotlight for the early detection of cancer. Solid tumors undergo angiogenesis and release various substances into the blood. In particular, circulating DNAs (ctDNAs), circulating RNAs (ctRNAs), proteins, vesicles such as exosomes, and circulating tumor cells (CTCs) have been found in the blood of cancer patients2,9. Although there are differences in the amount of these substances, they are consistently observed not only in the early stages but also in the later stages6,10. However, these individual differences are very high; for example, the amount of cell-free DNA (cfDNA) containing ctDNA is less than 1,000 ng, and the number of CTCs is less than 100 in 10 mL of whole blood from cancer patients11,12,13. Many studies have characterized cancer using these substances present in lesser amounts (i.e., cfDNA, ctDNA, and CTCs). To obtain accurate results, it is important to accurately separate small amounts of substances with high purity13,14. Conventional centrifugation methods are commonly used, but they are difficult to handle and have low purity depending on the user&#39;s skill. Since the discovery of CTCs, several separation techniques have been developed, such as centrifugation or density grade separation, immunobead, and microfluidic methods. Several containment techniques have been developed since the discovery of CTCs. However, these techniques are often limited when it is necessary to isolate cells from the various chips and membranes used to isolate them15. Also, the tagging methods require equipment such as FACS, and there are limits to the downstream process due to tagging contamination.
Recently, the use of liquid biopsies have increased, and various studies are being conducted for the early detection of cancer. Although this method is simple, there are still difficulties in downstream analysis, and various studies are attempting to overcome these difficulties16,17. In addition, many sites, including hospitals, require automated, reproducible, and high-purity methods that are convenient to use. Here, we have developed a lab-on-a-disc for the automated separation of substances from blood samples following a liquid biopsy. These devices are based on the principle of centrifugation, microfluidics, and pore-sized cell capture. There are three types of discs: LBx-1 can acquire plasma and buffy coat, while LBx-2 can acquire plasma and PBMC from whole blood with a volume of less than 10 mL; FAST-auto can also acquire CTCs using a membrane that is removable from the disc. Up to four of each disc can be used in one run. Above all, the advantage of this device and method is that it can obtain a variety of cancer-derived substances from the same sample using a small amount of blood. This means that the patient&#39;s blood only needs to be drawn once. In addition, it has the advantage of excluding errors due to differences in the blood sampling period. This platform is easy to use and provides accurate results for liquid biopsies and downstream applications. In this protocol, the usage of the device and cartridge is introduced.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% BSA (Bovine Serum Albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tube</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tube</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>4150 TapeStation System</td>
			<td>Agilent</td>
			<td>G2992AA</td>
			<td>Cell-free DNA Screen Tape (Agilent, 5067-5630), Cell-free DNA Sample Buffer (Agilent, 5067-5633)</td>
		</tr>
		<tr>
			<td>Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit&#160;</td>
			<td>Apostle</td>
			<td>A17622-250</td>
			<td>5 mL X 50 preps version</td>
		</tr>
		<tr>
			<td>BD Vacutainer blood collection tubes</td>
			<td>BD</td>
			<td>367525</td>
			<td>EDTA Blood Collection Tube (10 mL)</td>
		</tr>
		<tr>
			<td>BioViewCCBS</td>
			<td>Clinomics</td>
			<td></td>
			<td>BioView Clinomics-Customized Bioview System. Allegro Plus microscope-based customization equipment</td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (HI30), PE-Alexa Fluor 610</td>
			<td>Invitrogen</td>
			<td>MHCD4522</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST Auto cartridge</td>
			<td>Clinomics</td>
			<td>CLX-M3001</td>
			<td></td>
		</tr>
		<tr>
			<td>LBx-1 cartridge</td>
			<td>Clinomics</td>
			<td>CLX-M4101</td>
			<td></td>
		</tr>
		<tr>
			<td>LBx-2 cartridge</td>
			<td>Clinomics</td>
			<td>CLX-M4201</td>
			<td></td>
		</tr>
		<tr>
			<td>OPR-2000 instrument</td>
			<td>Clinomics</td>
			<td>CLX-I2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass</td>
			<td>Marienfeld Superior</td>
			<td>HSU-0101040</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag 2 Magnet Stand</td>
			<td>Thermo Fisher Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll Paque Solution</td>
			<td>GE healthcare</td>
			<td>17-1440-03</td>
			<td>density gradient solution</td>
		</tr>
		<tr>
			<td>Filter Tip, 10 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-10</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>Filter Tip, 200 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-200</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>Filter Tip, 100 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-100</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>Filter Tip, 1000 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-1000</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>FITC anti-human CD326 (EpCAM) Antibody</td>
			<td>BioLegend</td>
			<td>324204</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human Cytokeratin</td>
			<td>BD Biosciences</td>
			<td>347653</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution (35 wt. % in H2O)</td>
			<td>Sigma Aldrich</td>
			<td>433284</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech Science Wipers</td>
			<td>Yuhan-Kimberly</td>
			<td>41117</td>
			<td></td>
		</tr>
		<tr>
			<td>Latex glove</td>
			<td>Microflex</td>
			<td>63-754</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Bead Separation Rack</td>
			<td>V&#38;P Scientific</td>
			<td>VP 772F2M-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (0.5-10 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000020</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (2-20 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000038</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (10-100 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000046</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (20-200 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000054</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (100-1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000062</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI&#160;- Aqueous, Fluoroshield</td>
			<td>abcam</td>
			<td>ab104139</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Human IgG Control</td>
			<td>R&#38;D Systems</td>
			<td>1-001-A</td>
			<td></td>
		</tr>
		<tr>
			<td>OLYMPUS BX-UCB</td>
			<td>Olympus</td>
			<td>9217316</td>
			<td></td>
		</tr>
		<tr>
			<td>Pan Cytokeratin Monoclonal Antibody (AE1/AE3), Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>53-9003-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Saline Solution)</td>
			<td>Corning</td>
			<td>21-040CVC</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable Pipet Aid</td>
			<td>Drummond</td>
			<td>4-000-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Glass</td>
			<td>Marienfeld Superior</td>
			<td>HSU-1000612</td>
			<td></td>
		</tr>
		<tr>
			<td>StainTray Staining box</td>
			<td>Simport</td>
			<td>M920</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Serological Pipette (10 mL)</td>
			<td>SPL</td>
			<td>91010</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100 solution</td>
			<td>Sigma Aldrich</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN&#160;20</td>
			<td>Sigma Aldrich</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole Blood</td>
			<td></td>
			<td></td>
			<td>Stored at 4-8 &#176;C by collecting in EDTA or cfDNA stable tube : If the whole blood is insufficient in 9 mL, add PBS (phosphate buffered saline) as much as necessary.</td>
		</tr>
		<tr>
			<td>X-Cite 120Q (Fluorescence Lamp Illuminator)</td>
			<td>Excelitas</td>
			<td>010-00157</td>
			<td></td>
		</tr>
	</tbody>
</table>

automatic separation and collection of cancer-related substances from clinical samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

The goal of this technique is to easily and automatically isolate cancer-associated substances from whole blood. In particular, anyone can use this technique in all the suitable fields of research and analysis. The simultaneous and reproducible separation of multiple substances in a single blood sample is significant in liquid biopsies. The LBx-1 and LBx-2 discs are used for isolating plasma and buffy coat or PBMC from whole blood. Figure 1 shows the materials separated by the application of...

Watch this Scientific Journal Video about Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples at JoVE.com

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

automatic-separation-collection-cancer-related-substances-from

Research

JoVE Journal

Cancer Research

1.8K Views.  Clinomics Inc.. This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood. 

Video: Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

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 ...

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 ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

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.

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 ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

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.