Name: capture and release of viable circulating tumor cells from blood
Uploaded: 2016-10-28T14:00:00
Description: Watch this Scientific Journal Video about Capture and Release of Viable Circulating Tumor Cells from Blood at JoVE.com

We thank all the patients who have donated blood samples to support this work. We thank Drs. Guiseppe Giaconne, Ritesh Parajuli, and Marc E. Lippman for their assistance in clinical sample acquirement, and Drs. Carmen Gomez, Ralf Landgraf, Stephan Z&#252;chner, Toumy Guettouche, Diana Lopez for their insightful discussions. Zheng Ao thanks partial support and assistance from the Sheila and David Fuente Graduate Program in Cancer Biology, Sylvester Comprehensive Cancer Center.

Ethics Statement: To protect the rights of human subjects, blood samples were obtained following an informed consent under protocols approved by the University of Miami institutional review boards under IRB 20150020. 
NOTE: Blood to be filtered for CTC capture should be collected in an EDTA tube to prevent coagulation.
1. Coating the Microfilter with Poly (N-iso-propylacrylamide) (PIPAAm)
<ol>
	<li>Weigh out PIPAAm to prepare a 10% w/v solution in butanol. Mix using a vortex until the solution is clear.</li...

<ol>
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The process of capturing viable CTC from whole blood and releasing them from the microfilter is relatively straightforward; however a few critical points are worth mentioning. It is imperative, as with all cell culture that a sterile condition is maintained through the whole process. The initial step of coating the filter with PIPAAm is critical, as the basis for the technique of releasing the cells from the filter is based on exploiting PIPAAm&#39;s temperature responsive interfacial properties. To ensure the filter has...

Metastatic disease is responsible for most cancer deaths. Developing prognostic and companion diagnostic biomarker for metastasis is crucial in cancer management and treatment. Circulating tumor cells (CTC) play a central role in tumor dissemination and metastasis. Furthermore, being easily accessible as a &#39;liquid biopsy&#39; biomarker, CTC in cancer patients&#39; peripheral blood has been rising as a &#39;hotbed&#39; for cancer biomarker research. CTC have been well validated as a prognostic biomarker in various cancer settings, including breast, prostate and colorectal cancer1-3. However, recent advances in CTC field has indicated that the mere enumeration of these rare cells has limited clinical utility, as shown in interventional clinical trials4. Thus, there is an emerging need for technologies that allow for molecular and functional characterization of CTC. Currently, only few technologies exist that allow for non-antigen biased, viable capture and release of CTC, enabling robust downstream molecular and functional analysis5,6. Majority of these microfabricated devices are coupled to microfluidic platforms and thus have a limiting factor in the amount of blood that that can be processed, which ranges from 2-4 ml7-10 . CTC are rare events in a single tube of blood draw (7.5 ml), therefore further reducing the amount of blood that can be processed, greatly hinders the chances of capturing and isolating these cells of interest.
We have developed two types of Parylene C membrane microfilter devices for the capture of CTC which exploit the size differences between larger tumor cells and the smaller normal blood cells11,12. We have previously reported on the round pore enumeration filter and compared it with a FDA-approved platform, where the microfilter was shown to be superior in CTC capture efficiency for cancer patient blood samples13,14. However, a limitation of the round filter is the necessity to use a formaldehyde-based fixative prior to filtration. This process preserves the cells morphology while allowing them to withstand the shear stress and pressure during the filtration process. While enumeration and molecular studies can be performed on-chip13, the fixative impairs the ability to perform functional characterization. To address this limitation, we developed a slot pore filter that negates the necessity to fix cells prior to filtration (Figure 1). The slot pore geometry (6 &#181;m width x 40 &#181;m length slot pores) allows the tumor cells to be captured while only partially occluding a pore and thus still permitting free passage for other blood cells and alleviating the rise in pressure that would lead to cell damage and eventual bursting15,16 The slot pore cartridge is composed of 2 acrylic pieces which sandwich the slot pore filter between the top and bottom piece with Polydimethylsiloxane (PDMS) acting as a gasket to provide a leak proof seal14,15 (Figure 1).
While the capture efficiency of the slot pore filter is high, (Table 1), the captured CTC are bound to the Parylene C membrane by strong non-specific electrostatic interactions instead of extracellular matrix (ECM) mediated adhesion15. Methods such as reverse flow or the use of cell scrapers fail to effectively release the cells from the filter, or result in cell damage and cell death. We explored an unconventional use of PIPAAm to formulate a release strategy15. PIPAAm is a polymer that undergoes a reversible lower critical solution temperature (LCST) phase transition at a solution temperature of 32 &#176;C17. Traditionally, this property of PIPAAm has been widely explored for tissue engineering applications. Typically, cells are cultured on PIPAAm coated surfaces at 37 &#176;C when PIPAAm is hydrophobic. The cells can then be detached as a sheet when the culture temperature is shifted to below 32 &#176;C, where the PIPAAm coated surface becomes hydrated17,18. We exploited this thermal property by performing the filtration process at room temperature (below 32 &#176;C) and then enabling cell release by placing the filter in culture media maintained at 37 &#176;C. At this temperature, the PIPAAm polymer layer becomes hydrophobic, thereby releasing the electrostatically bound cells15 (Figure 1).
Although the temperature responsive method as well as other methods have been successfully implemented to achieve viable CTC capture and release19-21, one key potential drawback shared by these technologies reported is that they all employ an antigen-dependent principle for CTC capture. Antigen based CTC capture, as shown previously, may lead to biased CTC analysis11,14. For example, many affinity-based technologies employ antibody that binds EpCAM for CTC capture. However, CTC has been shown to express various levels of EpCAM, leading to omission of EpCAM low and EpCAM negative CTC by these technologies. Also, limitations may occur when CTC from non-epithelial origin is of interest, such as CTC in melanoma and sarcoma settings. Thus, a technology that allows for viable CTC capture and release without potential bias introduced by antigen based capture is highly desirable.
Importantly, the microfilter capture device is purely sized based and the release strategy is agnostic to the presence of certain surface markers. We believe that the employment of the PIPAAm coated microfilter will help to expand our understanding of the metastatic process, through providing the ability to effectively and efficiently capture and release CTC for downstream analyses. This may potentially expose new molecules for which novel targeted systemic therapies might be directed as well as provide a biomarker that can be monitored easily and aid in cancer patient management.

<table border="0" cellpadding="0" cellspacing="0" width="603">
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	<tbody>
		<tr height="106">
			<td height="106" style="height:107px;width:216px;">Slot Filter</td>
			<td style="width:101px;">Circulogix Inc.</td>
			<td style="width:119px;">MSF-01</td>
			<td style="width:167px;">Different size filters available based for filtration for CTC from blood or urine (www.circulogixinc.com)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">poly(N-iso-propylacrylamide) (PIPAAm)&#160;</td>
			<td style="width:101px;">Ploysciences Inc.</td>
			<td style="width:119px;">21458</td>
			<td style="width:167px;">Non-Hazardous. Store at room temp.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1-Butanol</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:119px;">B7906</td>
			<td style="width:167px;">Use in well ventilated area</td>
		</tr>
		<tr height="118">
			<td height="118" style="height:119px;width:216px;">Plastic Microscope Slides</td>
			<td style="width:101px;">Cole-Parmer</td>
			<td style="width:119px;">48510-30</td>
			<td style="width:167px;">Any plastic slides or alternatively any sort of square (Metal, Acrylic etc.) can be used if it will be bale to hold the 8mmx8mm filter square</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:216px;">Spin Coater</td>
			<td style="width:101px;">Specialty Coating Systems</td>
			<td style="width:119px;">SCS G3 Spin Coater</td>
			<td>Instrument</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:216px;">Polyimide Tape</td>
			<td style="width:101px;">Uline</td>
			<td style="width:119px;">S-7595</td>
			<td style="width:167px;">Polyimide is the generic name for Kapton Tape which can be purchased form multiple vendors (Amazon, Kaptontape.com)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">HBSS- Hank&#39;s Balanced Salt Solution</td>
			<td style="width:101px;">Gibco</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">1XPBS</td>
			<td style="width:101px;">Gibco</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:216px;">McCoy&#39;s</td>
			<td style="width:101px;">Gibco</td>
			<td style="width:119px;">16600-082</td>
			<td style="width:167px;">Warm in 37 &#8304;C water bath before use. McCoys was used for SKBr3 cells, if you use different cell lines or patient blood, please use media that would be optimal for that particular case</td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:216px;">Falcon Petri dishes 35x10 mm</td>
			<td style="width:101px;">VWR</td>
			<td style="width:119px;">25373-041</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:216px;">Microfilter Cassette</td>
			<td style="width:101px;">Circulogix Inc.</td>
			<td style="width:119px;">FC-01</td>
			<td style="width:167px;">Custom catridges are avilable based on filtration for CTC from blood or urine&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Syringe 20mL</td>
			<td style="width:101px;">BD Scientific</td>
			<td style="width:119px;">302830</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Syringe Pump</td>
			<td style="width:101px;">KD scientific&#160;</td>
			<td style="width:119px;">78-0100V</td>
			<td style="width:167px;">Any syringe pump capable of holding a 25mL syringe may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cellstar 50mL Centrifuge tube</td>
			<td style="width:101px;">VWR</td>
			<td style="width:119px;">82050-322</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Greiner Bio One 6 well plate</td>
			<td style="width:101px;">VWR</td>
			<td style="width:119px;">89131-688</td>
			<td style="width:167px;">Any brand can be used, as long as the surface is compatiable for cell adesion and not repellant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SKBR3 Cells</td>
			<td style="width:101px;">ATCC</td>
			<td style="width:119px;">HTB-30</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Live Dead Assay</td>
			<td style="width:101px;">Life Technologies</td>
			<td style="width:119px;">L3224</td>
			<td style="width:167px;">Any assay that can provide a reasonable analysis to evaluate live cells will work</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cell Culture Incubator</td>
			<td style="width:101px;">VWR</td>
			<td style="width:119px;">98000-368</td>
			<td style="width:167px;">Any incubator that can be used for cell culture will suffice</td>
		</tr>
	</tbody>
</table>

capture and release of viable circulating tumor cells from blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

Using healthy donors&#39; blood (obtained under a protocol approved by the University of Miami IRB 20150020 following an informed consent) spiked with cultured cancer cells, the thermoresponsive technique for release of viable circulating tumor cells (CTC),achieved capture, release and retrieval efficiency of 94% &#177; 9%, 82% &#177; 5% and 77% &#177; 5% respectively (Table 1)15. By comparison, the release and retrieval efficiency of uncoated filters were sign...

Watch this Scientific Journal Video about Capture and Release of Viable Circulating Tumor Cells from Blood at JoVE.com

Capture and Release of Viable Circulating Tumor Cells from Blood

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

The overall goal of this technique is to effectively capture viable circulating tumor cells or CTCs from whole blood. These CTCs can be placed into culture or be used for downstream analyses which require non fixed samples. This method can help answer key questions in the field of metastasis.It can also be used to follow disease progression, response to therapy and evaluate risk of recurrence. The advantage of this technique is the ability to perform label-free capture of viable CTCs from any solid tumor while remaining independent of biomarker expression. We first had the idea of releasing CTCs from the filter because we wanted to do downstream RNA analysis and culture of CTCs, instead of culturing them in-situ on the filter.To begin the experiment, weigh enough PIPAAm to prepare a 10%weight per volume solution in a 15 milliliter tube, containing butanol. Vortex the tube until the solution is clear. Next, cut plastic microscope slides into roughly 12 millimeter by 12 millimeter squares with a pair of scissors.Then, using a sharp pair of straight edge scissors, cut a slot-pore microfilter wafer into eight millimeter by eight millimeters squares and place these pieces directly onto the plastic squares. Using a polyimide film tape, secure the cut filters onto the plastic squares, only on the edge and the corner of the filter so that at least, seven millimeter by seven millimeter filter area is not covered by the tape. Place the microfilter that is secured on the plastic square onto the vacuum chuck of the spin coater.Program the spin coater to the following recipe. Then, with a standard plastic transfer pipette, dispense enough 10%weight per volume PIPAAm solution to completely cover the microfilter surface and start the spin coater. After the coating is completed, remove the PIPAAm coated microfilter from the spin coater and leave the filter attached to the plastic square during storage.Move on to filtration cassette assembly and rehydrate the PIPAAm coated microfilter at room temperature by placing the microfilter into a petri dish. Add 1X PBS until the microfilter is fully submerged. After the hydration step, release the filter from the plastic square by peeling or cutting the polyimide film tape from the filter.Next, assemble the microfilter into the filtration cassette by sandwiching the microfilter between the top and bottom acrylic piece along the PDMS pieces which act as a gasket. Then, clamp the acrylic cassette with clips to secure the filter, to provide a tight seal. Warm three milliliters of McCoy's cell culture medium to 37 degrees celsius.Add 7.5 milliliters of Hank's balanced salt solution or HBSS to the 7.5 milliliters of blood sample and aspirate this diluted blood into a 25 milliliter syringe. After identifying the top of the cassette, engage the filtration cassette with the syringe and place it onto the syringe pump. Set the syringe pump to a 75 milliliter per hour flow rate.Place a 50 milliliter tube at the bottom to collect the flow through and start the pump. After filtration is complete, disengage the filtration cassette, taking note which side is the top that has the cells caught in the PIPAAm coated surface. Aspirate one milliliter of the warm culture medium into a new syringe.Again, determine the top of the cassette and then re-engage the filtration cassette with the syringe containing the medium and engage the bottom end of the cassette so that the PIPAAm coated surface of the filter is facing away from the syringe. Then place the syringe back onto the syringe pump and hold the six well plate right under the filtration cassette. Set the flow rate and start the pump.Pass all the medium through the filter and collect the flow through in the petri dish. Wait for all the medium to pass through, then stop the pump and remove the syringe and filtration cassette from the pump. Next, disengage the filtration cassette from the syringe and open it to retrieve the filter from the cassette.Place the filter with the PIPAAm surface facing down into the same petri dish used to collect the reverse flow when releasing the cells from the pores. Add the rest of the warm medium and place the petri dish into a culture incubator, set at 37 degrees celsius. Once the filter has been retrieve from the cassette, it is critical to place it in the plate with the cells facing down and if needed, gently shake it in the media to ensure the cells are detached from the filter.After 24 hours in culture, carefully remove the microfilter using forceps and discard the filter. Gently resuspend the erythrocytes and peripheral blood mononuclear cells or PBMCs with a 1000 micro liter pipette. It is critical to be extremely gentle when resuspending the erythrocytes and PBMCs to avoid pipetting out the CTCs.Carefully remove the entire medium containing the resuspended erythrocytes and PBMCs while leaving behind the attached cancer cells and substitute the cells with fresh medium, pre-warmed to 37 degrees celsius. Proceed with CTC viability evaluation. Using healthy donors blood spiked with cultured cancer cells, the thermal responsive technique for release of viable circulating tumor cells or CTCs, achieved capture, release and retrieval efficiency of 94%82%and 77%respectively.The release and retrieval efficiency of uncoated filters were significantly lower. A live dead assay was performed to evaluate the cell viability before the spike into blood and after release. The cells remained viable post release and expanded in culture rapidly.SKBr-3 cells were retrieved from blood by the PIPAAm coated slot filter and plated on a 48 well plate. At 16 hours in a culture, tumour cells adhere to the culture plate along with a hypotonic erythrocytes and leukocytes which settled at the bottom of the plate. Post-wash non-adherent cells were removed only leaving adhering tumor cells on the plate.The same filters can be used for fixed cell capture, both for enumerating and molecular characterizations of CTCs to track progression of cancer and better cancer management. After its development, this technique paved the way for the researchers in the field of CTCs enabling functional characterization of these cells to explore future drug targets while taking a step towards personalized medicine.

Research

JoVE Journal

Cancer Research

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

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 ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...