Capture and Release of Viable Circulating Tumor Cells from Blood

Summary

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' blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

Abstract

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 °C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 °C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

Introduction

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 'liquid biopsy' biomarker, CTC in cancer patients' peripheral blood has been rising as a 'hotbed' for cancer biomarker research. CTC have been well validated as a prognostic biomarker in various cancer settings, including breast, prostate and colorectal cancer^1-3. 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 analysis^5,6. 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 ml^7-10 . 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 cells^11,12. 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 samples^13,14. 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 µm width x 40 µm 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 bursting^15,16 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 seal^14,15 (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 °C¹⁷. Traditionally, this property of PIPAAm has been widely explored for tissue engineering applications. Typically, cells are cultured on PIPAAm coated surfaces at 37 °C when PIPAAm is hydrophobic. The cells can then be detached as a sheet when the culture temperature is shifted to below 32 °C, where the PIPAAm coated surface becomes hydrated^17,18. We exploited this thermal property by performing the filtration process at room temperature (below 32 °C) and then enabling cell release by placing the filter in culture media maintained at 37 °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 release^19-21, 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 analysis^11,14. 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.

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Protocol

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)

1. Weigh out PIPAAm to prepare a 10% w/v solution in butanol. Mix using a vortex until the solution is clear.
2. Cut plastic microscope slides into roughly 12 mm x 12 mm squares either with a pair of scissors or sharp blade. Alternatively use a guillotine.
   NOTE: These squares will serve as a holder for the filters during the spin coating process.
3. Using a sharp pair of straight edge scissors, cut the slot pore microfilter wafer into 8 mm x 8 mm squares.
4. Using a polyimide film tape secure the cut filters onto the plastic squares previously cut in step 1.3. Apply the film only to the edge and the corner of the filter so that at least 7 mm X 7 mm of filter area is un-covered by the tape.

Figure 2: Representative Illustration of the Microfilter Set-up for PIPAAm Coating. 8 mm x 8 mm microfilter is positioned onto the 12 mm x 12 mm plastic square cut from a plastic microscope slide. Polyimide tape is used to secure the microfilter in place to spin coat the PIPAAm.Reproduced with permission from reference¹⁵. Please click here to view a larger version of this figure.

5. Place the microfilter that is secured on the plastic square, onto the vacuum chuck of the spin-coater.
6. Program the spin-coater to the following recipe: Start, 500 rpm for 10 sec, 6,000 rpm for 60 sec, 500 rpm for 10 sec, Stop.
7. Dispense enough 10% w/v PIPAAm solution (prepared in 1.1) to completely cover the microfilter surface using a standard plastic transfer pipette and start the spin coater.
8. Remove the PIPAAm coated microfilter from the spin-coater after the coating process is completed. Leave the filter attached to the plastic square during storage.
   NOTE: The coated-filter can be stored at room temperature for up to 3 months.

2. Assembly of Filtration Cassette with PIPAAm Coated Microfilter

1. Rehydrate PIPAAm coated microfilter at room temperature by placing the microfilter still secured on the plastic square into a petri dish. Add 1x PBS until the microfilter is fully submerged and let stand for 5 min.
2. After the hydration step, release the filter from the plastic square by cutting the polyimide film tape using a pair of scissor.
   NOTE: Take note of which side of the filter has the PIPAAm coating.
3. Assemble the microfilter into the filtration cassette, by sandwiching the microfilter between the top and bottom acrylic piece along with the 2 Polydimethylsiloxane (PDMS) pieces acting as a gasket/seal. Clamp the acrylic cassette with clips to secure the filter and provide a tight seal.
   NOTE: The PIPAAm coating should be facing upwards, so as the sample is filtered through, the cells will be captured on PIPAAm coated surface of the filter (Figure 1).

3. Filtration of Blood Sample with Capture and Release of CTC from the Microfilter

1. As each syringe pump brand has its own user interface, refer to the user manual of the pump and program the syringe pump to flow at a rate of 75ml/hr and set the volume to 20 ml.
2. Warm 3 ml of McCoy's (commercially available) cell culture media (prepared by adding in 10% Fetal Bovine Serum and 1% Penicillin and Streptomycin) to 37 °C.
   NOTE: The media choice will be based on the patient's cancer type or else if this is a model system experiment where cultured cells are to be spiked into healthy donor blood, use the media the cells are normally cultured in. If unsure, please refer to culturing guide/method provided by the company the cells were purchased from.
3. Add 7.5 ml of commercially available Hank's Balanced Salt Solution (HBSS) to the 7.5 ml of blood sample and aspirate this diluted blood into a 25 ml syringe.
   NOTE: Adjust the volume of HBSS so that the blood sample is diluted with equal volume of 1:1 with HBSS. E.g. if 10 ml of blood is used, then mix with equal volume of 10 ml HBSS to achieve the 1:1 dilution.
4. Engage the filtration cassette with the syringe and place it onto the syringe pump. Place a 50 ml tube at bottom to collect the flow through and start the pump.
   NOTE: The filtration should take roughly 10- 12 min. All filtration steps are to be carried out at Normal Room Temperature (20-25 °C) including step 3.8
5. After filtration is complete, disengage the filtration cassette taking note which side is the top that has the cells caught on the PIPAAm-coated surface. Aspirate 1 ml of the warm culture media into a new syringe.
6. Re-engage the filtration cassette with the syringe containing the media, but at this time engage the bottom end of the cassette so that the PIPAAm coated-surface of the filter is facing away from the syringe.
   NOTE: This will be to release any cells that may be embedded into the slot pores by applying gentle reverse flow.
7. Place the syringe back onto the syringe pump and hold a small petri dish or 6-well plate right under the filtration cassette. Set the flow rate to 100 ml/hr and start the pump.
8. Pass all the media through the filter and collect the flow-through in the petri dish. Wait for all the media to pass through and then stop the pump and remove the syringe and filtration cassette from the pump.
9. 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 media and place the petri dish into a culture incubator set at 37 °C.
10. After 30 min all the cells will be released from the filter. However, leave filter in the petri dish for up to 24 hours to ensure all cells detach.
    NOTE: Alternatively, the released cells can be collected into a microcentrifuge tube to perform further downstream analysis such as PCR, ELISA, Western Blot to name a few examples.
11. After 24 hr in culture, carefully remove the microfilter using forceps.
    NOTE: The filter can be discarded at this time as the cells have been released from it.
12. Gently pipette the culture medium up and down using a 1,000 µl pipette, to re-suspend the erythrocytes and peripheral blood mononuclear cell (PBMC) that are caught on the filter and released into the plate with the CTC. Perform this action carefully and gently to avoid detaching the loosely-attached cancer cells. Carefully remove the entire medium containing the re-suspended erythrocytes and PBMC while leaving behind the attached-cancer cells and substitute with fresh medium pre-warmed to 37 °C.
    NOTE: This step can be repeated once if excessive erythrocytes are present after one wash.

4. CTC Viability Evaluation

1. Evaluate cultured CTC viability using a Live/Dead Assay⁸.
   NOTE: Numerous Live/Dead assays are available commercially. Following the manufacturer's protocol is highly advised. Please refer to the materials/reagents list for the commercial assay kit used for this experiment.

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Results

Using healthy donors' 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% ± 9%, 82% ± 5% and 77% ± 5% respectively (Table 1)¹⁵. By comparison, the release and retrieval efficiency of uncoated filters were sign...

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Discussion

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's temperature responsive interfacial properties. To ensure the filter has...

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Materials

Name	Company	Catalog Number	Comments
Slot Filter	Circulogix Inc.	MSF-01	Different size filters available based for filtration for CTC from blood or urine (www.circulogixinc.com)
poly(N-iso-propylacrylamide) (PIPAAm)	Ploysciences Inc.	21458	Non-Hazardous. Store at room temp.
1-Butanol	Sigma Aldrich	B7906	Use in well ventilated area
Plastic Microscope Slides	Cole-Parmer	48510-30	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
Spin Coater	Specialty Coating Systems	SCS G3 Spin Coater	Instrument
Polyimide Tape	Uline	S-7595	Polyimide is the generic name for Kapton Tape which can be purchased form multiple vendors (Amazon, Kaptontape.com)
HBSS- Hank's Balanced Salt Solution	Gibco	14025-092
1x PBS	Gibco	10010-023
Falcon Petri dishes 35 x 10 mm	VWR	25373-041
Microfilter Cassette	Circulogix Inc.	FC-01	Custom catridges are avilable based on filtration for CTC from blood or urine
Syringe 20 ml	BD Scientific	302830
Syringe Pump	KD scientific	78-0100V	Any syringe pump capable of holding a 25 ml syringe may be used
Cellstar 50 ml Centrifuge tube	VWR	82050-322
Greiner Bio One 6 well plate	VWR	89131-688	Any brand can be used, as long as the surface is compatiable for cell adesion and not repellant
SKBR3 Cells	ATCC	HTB-30
Live Dead Assay	Life Technologies	L3224	Any assay that can provide a reasonable analysis to evaluate live cells will work
Cell Culture Incubator	VWR	98000-368	Any incubator that can be used for cell culture will suffice