Name: Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry (Video) | JoVE Journal
Uploaded: 2011-11-25T13:00:00.000+00:00
Duration: 9 min 52 s
Description: Learn photoacoustic flowmetry for detecting circulating melanoma cells in blood samples. This label-free method enables rapid isolation of cancer cells before secondary tumor formation for improved early diagnosis.

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5 minutes

We acknowledge the support of the Department of Biological Engineering and the Christopher S. Bond Life Sciences Center at the University of Missouri. We acknowledge grant support from Missouri Life Sciences Research Board 09-1034 and NIH R21CA139186-0. We also thank the Life Sciences Undergraduate Research Opportunity Program at the University of Missouri, the University of Missouri Molecular Cytology Core, and the University of Missouri College of Engineering for financial support.

1. Blood sample preparation
<ol>
	<li>Draw approximately 10 mL of whole blood from a Stage IV Cancer patient into two 5 mL vacutainers.</li>
	<li>Centrifuge the tubes for ten minutes at 3,000 revolutions per minute (rpm).</li>
	<li>The blood sample will separate into three different layers: the bottom layer comprises the erythrocytes, the middle layer, also called the buffy coat, contains leukocytes and melanoma cells, and the top layer contains plasma and platelets. Remove as much plasma as possible without disturbing the buffy coat.</li>
	<li>Place 250 μL of Histopaque 1077 in two 1 mL Wintrobe tubes. </li>
	<li>Using a 9″ Pasteur Pipette, extract the remaining plasma, the entire buffy coat, and even the top layer of erythrocytes, making sure to collect &lt; 500 μL.</li>
	<li>Place the solution atop the Histopaque layer and centrifuge the Wintrobe tubes in 15 mL conical tubes for 10 minutes at 3000 rpm. </li>
	<li>The blood will again separate, but this time the erythrocytes will be separated by the Histopaque, allowing for easy extraction of the buffy coat. Use a 9″ Pasteur Pipette to obtain the entire buffy coat and place it in a 2 mL eppendorf tube. </li>
	<li>Suspend the buffy coat in 1.5 mL of PBS with 2% v/v Tween 20. </li>
</ol>
2. Flow Chamber Construction
<ol>
	<li>Obtain an acrylic cylinder, 20 mm outer diameter, 17 mm inner diameter, and 8 mm tall. Drill three 5 mm diameter holes through the side of the cylinder at 90 degree angles from each other. Cut three pieces of polymer tubing (1.6 mm inner diameter, 5 mm outer diameter), and place them into the drilled holes.</li>
	<li>Stretch Parafilm across the bottom of the acrylic ring to make a water tight seal. The parafilm forms a shallow bowl with the acrylic ring and will hold the acrylamide solution that will form a flow chamber.</li>
	<li>Suspend a 1.6 mm diameter wire through the pieces of tubing that are across from each other, making sure that the wire alone is visible through the middle of the ring. Fill the third piece of tubing with a 1.6 mm diameter wire and position the tube 1 mm away from the suspended wire. These wires prevent acrylamide from entering the tubing. After the acrylamide solidifies, the wires will be extracted, leaving a cylindrical flow path and a place to position an optical fiber focuses onto the flow path.</li>
	<li>Add 5 mL of prepared acrylamide [20 g acrylamide (Sigma Aldrich), 0.7 g bisacrylamide (Sigma Aldrich), 100 mL of distilled water] to a small beaker. Measure out 0.02 g of ammonium persulfate (Sigma) and add it to the beaker. Mix with a stir bar for 3 minutes. Add 20 μL tetramethylethylenediamene (Fisher Scientific) to the beaker. Quickly pour the mixture into the acrylic ring, filling to the top. After a few seconds, the solution will gel, making a flow chamber mold. Carefully pull the wires out, making sure not to disturb the acrylamide gel. The flow chamber is now ready for use.</li>
</ol>
3. Photoacoustic flowmetry set-up
<ol>
	<li>Acoustically couple a polyvinylidene fluoride piezoelectric acoustic transducer (Panametrics) to a flow chamber using ultrasound gel directly above the flow path.</li>
	<li>Using a Bayonet Neill-Concelman (BNC) cable, connect the transducer to the input of a RITEC Broadband Receiver-640A (lowpass filter: out, highpass filter: 1 MHz, imput impedance: 50 Ohms, amplification: 35 dB). Connect the output of the filter to the oscilloscope (Tektronix TDS 2024B). To trigger the oscilloscope, a photodiode (Thorlabs) is set up near the transducer and is plugged into the oscilloscope using a BNC cable.</li>
	<li>A t-connector (Small Parts) is attached to one arm of the flow chamber. Two syringe pumps (Braintree Scientific) are connected to the other branches of the connector. One syringe contains air, while the other will contain the cell suspension prepared in part 1. Set the flow rates to 100 μL/minute.</li>
	<li>Extract the small tubing of the flow chamber until a small pocket of air forms, and insert a 1 mm diameter core optical fiber with a 0.37 numerical aperture in that slot. Fill the pocket with water to reduce interface signals and increase the amount of laser light applied to the flow path. Due to the numerical aperture of the optical fiber and short distance to the flow path, the laser will only irradiate one slug of sample at any given time, ensuring that the sample directly in front of the laser is the same sample creating photoacoustic signals. The laser energy should be approximately 28 mJ/cm2 with a spot size of 7 mm2.</li>
	<li>Turn both syringe pumps on. When the two immiscible fluids reach the t-junction they will be partitioned into homogeneous slugs and flow past the transducer.</li>
	<li>Irradiate the flowing sample with a nanosecond pulsed laser to generate photoacoustic waves in the melanoma cells. As melanin is a broadband optical absorber, any visible laser wavelength can be used. </li>
	<li>If a signal appears on the oscilloscope when a slug is atop the transducer, the slug contains melanoma, and should be tracked through the system until dropping into a collection vial.</li>
</ol>
4. Immunocytochemistry
<ol>
	<li>Take the captured slugs to a Cytospin 4 Centrifuge (Thermo Scientific) in order to fix them to glass slides. Place glass slides into Cytofunnels (Shandon EZ Single Cytofunnel with Brown Filter Card Thermo Scientific) and load them into the Cytospin 4 Centrifuge. Put 100 μL of 1% Bovine Serum Albumin (Sigma) in PBS into each funnel and then centrifuge at 600 rpm for 3 minutes to help the captured cells stick to the glass slides.</li>
	<li>After centrifugation, add the cell samples to the Cytofunnels and then centrifuge at 600 rpm for 3 minutes. Then spray the slides with an ethanol based fixative.</li>
	<li>Wash the slides in PBS for 10 minutes twice.</li>
	<li>To block nonspecific binding, incubate each slide with a mixture of 10 μL of Goat Serum (Sigma) and 90 μL of PBS for 1 hour at room temperature.</li>
	<li>After the incubation, decant the slides and incubate with the primary antibody solution for 1 hour in a humidified chamber at room temperature. Each slide should contain 1% MART-1 antibody raised in rabbit, 1% CD-45 antibody raised in mouse, 10% Goat serum, and 89% PBS.</li>
	<li>After incubation, wash the slides in PBS for 10 minutes three times.</li>
	<li>The slides are then incubated in a secondary antibody solution, which has fluorophore bound to the antibodies. The mixture contains 0.05% goat anti-rabbit antibody, 0.05% goat anti-mouse antibody, 0.1% goat serum, and 99% PBS. The slides are incubated in the dark for 1 hour at room temperature.</li>
	<li>After incubation, decant the slides wash in PBS for 5 minutes three times.</li>
	<li>To stain for a nucleus, incubate the cells with 0.1 μg/mL of DAPI (Invitrogen) for 1 minute. Then decant the slides and wash in PBS.</li>
	<li>Mount a coverslip over the cell sample using 20 μL of a resin-based mounting media per slide. Seal the coverslip with clear nail polish to preserve the slide. Now the slides are ready to be imaged with a fluorescent microscope.</li>
</ol>
5. Representative Results: 
<img src="/files/ftp_upload/3559/3559fig1.jpg" alt="Cell imaging and electrical signal graphs; white blood cells, melanoma cells; microscopy, voltmeter." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. 
The photoacoustic waveforms from white blood cells and melanoma cells are shown here. The white blood cells, having no inherent pigmentation, produce no photoacoustic waves and manifest as a flat line of electronic noise (left). The pigmented melanoma cells produces a robust photoacoustic wave (right).
<img src="/files/ftp_upload/3559/3559fig2.jpg" alt="Fluorescence microscopy image of stained cells; blue nuclei, green cytoplasm analysis." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2. 
After immunocytochemical staining, cultured melanoma cells show DAPI signals in blue while MART1 is highlighted in green.
<img src="/files/ftp_upload/3559/3559fig3.jpg" alt="Fluorescent microscopy image; cell staining; protein localization; immune response study; lab analysis." data-altupdatedat="1787630408000" data-alt="Figure 3"> Figure 3. 
Overlaying images for DAPI and MART1, as in Figure 2, with CD45 as an indicator of leukocytes, this figure shows melanoma cells among several white blood cells.

<ol><li>Pantel, K., Brakenhoff, R., Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Detection clinical relevance and specific biological properties of disseminating tumour cells.</a> Nature Reviews Cancer. 8, 329-340 (2008).</li>
<li>Pantel, K., Brakenhoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissecting+the+metastatic+cascade%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Dissecting the metastatic cascade.</a> Nature Reviews Cancer. 4, 448-456 (2004).</li>
<li>Yu, M., Stott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+tumor+cells%3A+approaches+to+isolation+and+characterization%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of Cell Biology. 192, 373-373 (2011).</li>
<li>Detection of circulating melanoma cells in human blood using photoacoustic flowmetry. Weight, R. M., Dale, P. S., Viator, J. Annual International Conference of the IEEE, 2009, , IEEE. 106-109 (1985).</li>
<li>Weight, R. M., Dale, P. S., Lisle, A., Caldwell, C., Viator, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photoacoustic+detection+of+metastatic+melanoma+cells+in+the+human+circulatory+system%5BTitle%5D)+AND+%22Opt.+Lett%22%5BJournal%5D)">Photoacoustic detection of metastatic melanoma cells in the human circulatory system.</a> Opt. Lett. 31, 2998-3000 (2006).</li>
<li>Gupta, A., Kumar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+geometry+on+droplet+formation+in+the+squeezing+regime+in+a+microfluidic+T-junction%5BTitle%5D)+AND+%22Microfluidics+and+Nanofluidics%22%5BJournal%5D)">Effect of geometry on droplet formation in the squeezing regime in a microfluidic T-junction.</a> Microfluidics and Nanofluidics. 8, 799-812 (2010).</li>
<li>Mocellin, S., Hoon, D., Ambrosi, A., Nitti, D., Rossi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+prognostic+value+of+circulating+tumor+cells+in+patients+with+melanoma%3A+a+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">The prognostic value of circulating tumor cells in patients with melanoma: a systematic review and meta-analysis.</a> Clinical Cancer Research. 12, 4605-4605 (2006).</li>
<li>Cristofanilli, M., Budd, G., Ellis, M., Stopeck, A., Matera, J., Miller, M., Reuben, J., Doyle, G., Allard, W., Terstappen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+tumor+cells%2C+disease+progression%2C+and+survival+in+metastatic+breast+cancer%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Circulating tumor cells, disease progression, and survival in metastatic breast cancer.</a> New England Journal of Medicine. 351, 781-781 (2004).</li>
<li>Cristofanilli, M., Hayes, D., Budd, G., Ellis, M., Stopeck, A., Reuben, J., Doyle, G., Matera, J., Allard, W., Miller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+tumor+cells%3A+a+novel+prognostic+factor+for+newly+diagnosed+metastatic+breast+cancer%5BTitle%5D)+AND+%22Journal+of+Clinical+Oncology%22%5BJournal%5D)">Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer.</a> Journal of Clinical Oncology. 23, 1420-1420 (2005).</li>
<li>Ghossein, R., Scher, H., Gerald, W., Kelly, W., Curley, T., Amsterdam, A., Zhang, Z., Rosai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+circulating+tumor+cells+in+patients+with+localized+and+metastatic+prostatic+carcinoma%3A+clinical+implications%5BTitle%5D)+AND+%22Journal+of+Clinical+Oncology%22%5BJournal%5D)">Detection of circulating tumor cells in patients with localized and metastatic prostatic carcinoma: clinical implications.</a> Journal of Clinical Oncology. 13, 1195-1195 (1995).</li>
<li>Loberg, R., Fridman, Y., Pienta, B., Keller, E., McCauley, L., Taichman, R., Pienta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+and+isolation+of+circulating+tumor+cells+in+urologic+cancers%3A+a+review%5BTitle%5D)+AND+%22Neoplasia%22%5BJournal%5D)">Detection and isolation of circulating tumor cells in urologic cancers: a review.</a> Neoplasia. 6, 302-302 (2004).</li>
<li>Viator, J. A., Gupta, S. K., Goldschmidt, B. S., Bhattacharyya, K., Raghuraman, R., Shukla, R., Dale, P. S., Boote, E., Katti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+gold+nanoparticle+enhanced+prostate+cancer+cells+using+photoacoustic+flowmetry+with+optical+reflectance%5BTitle%5D)+AND+%22J.+Biomed.+Nanotech%22%5BJournal%5D)">Detection of gold nanoparticle enhanced prostate cancer cells using photoacoustic flowmetry with optical reflectance.</a> J. Biomed. Nanotech. 6, 187-191 (2010).</li>
</ol>

John A. Viator is founder and president of Viator Technologies Inc, a company formed to commercialize photoacoustic technologies for the improvement of human health.

Detecting CTCs is still a research intensive field with very few clinical applications due to the difficult problem of isolating the rare tumor cells. Many other techniques are being evaluated for CTC detection, including RT-PCR, microfluidic cell capture, immunomagnetic capture, and other methods8-12. However, photoacoustic detection of CMCs shows promise as it is label free and provides a rapid and accurate means to capture small, light absorbing particles. 
The protocol described to isolate white blood cells is highly efficient, yet a low number of red blood cells exist in the sample. These rouge contaminating cells also absorb laser light, but at a greatly decreased level. To ensure the red blood cells do not interfere with our melanoma detection system, five healthy patient samples were introduced through the system and none gave a signal, indicating that red blood cells are present at low enough concentrations to be disregarded. 
A series of concentration studies have been conducted using cultured melanoma cells and the photoacoustic flow system has proven sensitive down to 1 cell/&mu;L (unpublished data). These studies are on going and will soon include trials using cancer patient samples.
This photoacoustic technique is also being evaluated for use in non-pigmented CTCs, such as breast and prostate cancer by attaching exogenous chromophores. We have performed preliminary work using gold nanoparticles on cancer cells13. These tests have shown that nanoparticles can provide the necessary optical absorption to generate photoacoustic waves. The remaining technical challenge is to attach such particles to cancer cells selectively.

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>ABCD1234</td><td></td></tr></tbody></table>

detection and isolation of circulating melanoma cells using photoacoustic flowmetry

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

The goal of the following experiment is to detect the presence of cancer before formation of secondary tumors. This is achieved by first obtaining and separating blood samples from stage four melanoma patients. As a second step, the blood is introduced into a photoacoustic flow chamber, which allows detection and isolation of melanoma cells.Next, the captured cells are imaged to confirm the presence of detected melanoma. The results show the system's ability to accurately and rapidly detect and isolate melanoma cells based on photoacoustic wave forms and cell imaging techniques. The main advantage of our technique over existing methods that detect circulating melanoma cells, such as R-T-P-C-R, biomes Magnetic Cell Sorting and other methods, is that our method is label free, and it allows for capture of circulating melanoma cells without altering their surface or securing them to a substrate.To prepare a sample for metastatic cancer detection begin by drawing approximately 10 milliliters of whole blood from a stage four cancer patient In two five milliliter vacutainer centrifuge, the tubes for 10 minutes. At 3000 RPM, the blood sample will separate into three different layers. The bottom layer comprises the erythrocytes.The middle layer, also called the buffy coat, contains leukocytes and melanoma cells, and the top layer contains plasma and platelets. Remove as much plasma as possible without disturbing the Buffy coat. Add 250 microliters of his opaque 10 77 to two milliliter troop tubes.Using a nine inch pastel pipette extract the remaining plasma, the entire Buffy coat, and the top layer of erythrocytes. Making sure to collect less than 500 microliters. Transfer the solution into the previously prepared wind Trobe tubes.The blood will again separate, but this time the erythrocytes will be separated by the hispa, allowing for easy extraction of the Buffy coat. Use a nine inch Pesa pipette to isolate the entire Buffy coat and place it in a two milliliter eend orph tube. Then suspend it in 1.5 milliliter of PBS with 2%volume per volume between 20.To construct a flow chamber, start with an eight millimeter long acrylic cylinder with a 20 millimeter outer diameter and a 17 millimeter in a diameter. Next drill, three five millimeter diameter holes through the side of the cylinder at 90 degree angles from each other. Then cut three pieces of polymer tubing and place them into the drilled holes.Stretch para film across the bottom of the acrylic ring to make a watertight seal. The para film will form a shallow bowl with the acrylic ring and will hold the acrylamide solution that will form a flow chamber. Suspend a 1.6 millimeter diameter wire through the pieces of tubing directly across from one another, making sure that the wire alone is visible through the middle of the ring into the third piece of tubing.Insert a 1.6 millimeter diameter wire and position the tube one millimeter away from the suspended wire. These wires prevent acrylamide from entering the tubing. Prepare five milliliters of acrylamide.Then quickly pour the mixture into the acrylic ring, filling it to the top. After a few seconds, the solution will gel. Making a flow chamber mold carefully pull the wires out while making sure not to disturb the acrylamide gel, leaving a cylindrical flow path and a place to position an optical fiber focused onto the flow path.The flow chamber is now ready for use using ultrasound gel acoustically. Couple a polyvinyl ledine fluoride piso electric acoustic transducer to a flow chamber directly above the flow path. Using a bayonet neo cons or BNC cable, connect the transducer to the input of a broadband receiver.Connect the output of the filter to the oscilloscope to trigger the oscilloscope. Set up a photo diode near the transducer and use a B NNC cable to plug it into the oscilloscope. Extract the small tubing of the flow chamber until a small pocket of air forms and couple it with the transducer.Then insert the optical fiber and fill the pocket with water to reduce interface signals and increase the amount of laser light applied to the flow path. Due to the numerical aperture of the optical fiber and the short distance to the flow path. The laser will only irradiate one slug of sample at any given time, ensuring that the sample directly in front of the laser is the same sample creating photo acoustic signals.Next, attach a T connector to one arm of the flow chamber and two syringe pumps to the other branches of the connector. One syringe contains air while the other will contain the previously prepared cell suspension. Then start both syringe pumps.When the two admissible fluids reach the T junction, they'll be partitioned into homogeneous slugs and flow past the transducer. Irradiate the flowing sample with a nanosecond pulse laser to generate photoacoustic waves in the melanoma cells. As melanin is a broadband optical absorber, any visible laser wavelength can be used.A signal appearing on the oscilloscope when a slug is atop the transducer indicates that the slug contains melanoma. Track it through the system before dropping it into a collection vial. To prepare samples for immuno cyto chemistry, place glass slides into cyto funnels and load them into the cyto spin for centrifuge to help the captured cells stick to the glass slides.Put 100 microliters of 1%BSA in PBS into each funnel. Then centrifuge at 600 RPM for three minutes. After centrifugation, add the cell samples for the CyTOF funnels, then centrifuge at 600 RPM for three minutes.Spray the slides with an ethanol based fixative. Then wash the slides in PBS for 10 minutes twice to block non-specific binding. Incubate each slide with a mixture of 10 microliters of goat serum and 90 microliters of PBS for one hour at room temperature.After the incubation, decant the slides and add primary antibody solution. Each slide should contain 1%rabbit mark, one 1%mouse CD 45 and 10%goat serum in PBS incubate for one hour in a humidified chamber at room temperature. Next, wash the slides in PBS for 10 minutes three times.Then incubate the slides in the appropriate fluorescent secondary antibodies in the dark for one hour at room temperature, decant the slides. Then wash them in PBS for five minutes, three times to stain the nuclei, incubate the cells with 0.1 micrograms per milliliter of DPI for one minute. Then decant the slides and wash in PBS.Finally, using a resin based mounting medium mount a cover slip over the cell sample seal the cover slip with clear nail polish and image with a fluorescent microscope. The photoacoustic wave forms from white blood cells and melanoma cells are shown here on the left. The white blood cells having no inherent pigmentation produce no photoacoustic waves and manifest as a flat line of electronic noise on the right.However, the pigmented melanoma cells produce a robust photoacoustic wave. After immuno cyto, chemical staining, cultured melanoma cells show dappy signals in blue, one marked one is highlighted in green overlaying images for DPI and mart one with CD 45 as an indicator of leukocytes. This figure shows melanoma cells among several white blood cells After its development.Our technique will pave the way for researchers in the field of cancer detection to explore the phenomenon of metastasis to both improve cancer detection and ultimately cure its fatal spread.

detection-isolation-circulating-melanoma-cells-using-photoacoustic

Research

JoVE Journal

Bioengineering

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Melanoma Stem Cell Isolation: A Method to Obtain CD133 Positive and Negative Melanoma Cells Using Magnetic-activated Cell Sorting

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In Vitro Photoacoustic Flow Cytometry: A Non-invasive Flow Photoacoustic Technique to Detect Nanoparticle-bearing Circulating Ovarian Cancer Cells

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Photoacoustic Monitoring of Dye Movement in Cortical Blood Vessels

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Here we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. This technique evanescent field-based photoacoustics can be used to create a photoacoustic metrology system to estimate materials' thicknesses, bulk and thin film refractive indices, and explore their optical properties.

Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

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This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

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Learn photoacoustic flowmetry for detecting circulating melanoma cells in blood samples. This label-free method enables rapid isolation of cancer cells before secondary tumor formation for improved early diagnosis.

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

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