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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

Introduction

Ovarian cancer is one of the deadliest gynecological malignancies and resulted in an estimated 184,800 deaths worldwide in 20181. Multiple studies have shown the correlation between ovarian cancer progression (i.e., metastasis) and the presence of CTCs2,3,4. The most common method for detection and isolation of CTCs utilizes the Cellsearch system, which targets the EpCam receptor5. EpCam expression, however, is downregulated in epithelial to mesenchymal transition, which has been implicated in cancer metastasis6. Despite advances, current clinical technologies still suffer from low accuracy, high cost, and complexity. Due to these drawbacks, new technologies for the discovery and enumeration of ovarian CTCs has become an important area for research.

Recently, PAFC emerged as an effective method for the noninvasive detection of cancer cells, analysis of nanomaterials, and identification of bacteria7,8,9. PAFC differs from traditional fluorescence flow cytometry by detecting analytes in flow by utilizing photoacoustics. The photoacoustic effect is generated when laser light is absorbed by a material that causes thermoelastic expansion, producing an acoustic wave that can be detected by an ultrasound transducer10,11. Advantages of PAFC over traditional flow cytometry methods include simplicity, ease of translation to clinical settings, and the detection of CTCs at unprecedented depths in patient samples12,13. Recent studies have utilized PAFC systems for the detection of cells using endogenous and exogenous contrast14,15. Near infrared (NIR) light-absorbing contrast agents such as indocyanine green dye, and metal NPs (e.g., gold and CuS) have been used for the selective labeling of cells and tissues in combination with photoacoustic imaging16,17,18. Due to the improved penetration depth of NIR light within biological tissues, photoacoustic detection of absorbers can be performed at greater depths for clinical applications. Because of its great potential for use in the clinic, the combination of targeted NIR contrast agents with PAFC has generated considerable interest for the detection of CTCs.

PAFC in combination with targeted contrast agents provides an improved approach for high-throughput analysis of patient samples with enhanced accuracy and targeted detection of CTCs. One of the principal detection strategies for CTCs is the specific targeting of membrane proteins present on the cell of interest. One notable characteristic of ovarian CTCs is the overexpression of folate receptors located on their outer membrane19. Folate receptor targeting is an ideal strategy for the identification of ovarian CTCs in blood because endogenous cells, which have higher expression of folic acid receptors, are generally luminal and have limited exposure to the bloodstream20. Copper sulfide NPs (CuS NPs) have recently been recognized for their ability to target folate receptors expressed on cancerous cells21. Combined with their biocompatibility, ease of synthesis, and absorption deep in the NIR, these NP contrast agents make an ideal targeting strategy for the detection of ovarian CTCs utilizing PAFC.

This work describes the preparation of FA-CuS NPs and their use for the detection of ovarian cancer cells in a photoacoustic flow system. CuS NPs are modified with folic acid to specifically target ovarian CTCs and emit a photoacoustic signal when stimulated with a 1,053 nm laser. The results indicate the successful detection of ovarian cancer cells incubated with these photoacoustic contrast agents within the PAFC system. These results show detection of ovarian cancer cells down to concentrations of 1 cell/µL, and fluorescence microscopy confirms successful uptake of these particles by SKOV-3 ovarian cancer cells22. This work provides a detailed description of the FA-CuS NPs synthesis, preparation of samples for fluorescence microscopy, construction of the photoacoustic flow system, and the photoacoustic detection of ovarian cancer cells. The presented method shows successful identification of ovarian CTCs in flow utilizing FA-CuS NPs. Future work will focus on the clinical application of this technology towards the early detection of ovarian cancer metastasis.

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Protocol

1. Nanoparticle Synthesis and Functionalization

NOTE: Synthesis of the FA-CuS NPs is achieved using a one pot synthesis method adapted from a previously published protocol21.
CAUTION: All synthesis should occur in a ventilated chemical fume hood.

  1. Prior to synthesis, filter approximately 300 mL of deionized (DI) water though a 0.2 µm sterile filter.
  2. Clean a 250 mL glass round bottom flask with a detergent solution and rinse with DI water. Add 0.0134 g of CuCl2 into 100 mL of DI water to create a 1 mM solution.
  3. Add 0.015 g of folic acid (FA) to the CuCl2 solution and stir for ~5 min using a magnetic stir bar.
  4. Add Na2S·9H2O (0.024 g in 100 µL DI water) over approximately 10 s to the reaction mixture utilizing a 200 µL pipette.
    NOTE: Upon addition of the Na2S·9H2O, the solution will change color from a light yellow to a dark brown.
  5. Cap the reaction and place in an oil bath, set to 90 °C, and continue stirring with a magnetic stir bar. After approximately 15 min, or when the oil bath has reached 85−90 °C, allow the reaction to proceed for an additional hour. Your mixture should gradually turn to a dark green color.
    NOTE: Make sure to vent the system while heating the reaction mixture to avoid pressure buildup.
  6. Remove the reaction vessel from the oil bath and briefly cool at room temperature for approximately 10 to 15 min before transferring to an ice bath.
  7. Once the reaction mixture has cooled below 20 °C, adjust the pH to 10 utilizing 1M NaOH to dissolve the remaining folic acid into solution.
  8. Purify the FA-CuS reaction mixture using a 30 kDa centrifugation column. Add solution in 15 mL batches to the column and centrifuge at 3,082 x g for 15 min.
  9. Once all of the reaction mixture has been concentrated, recombine the concentrated fractions and wash 4x with 15 mL of pH 10 NaOH in the 30 kDa centrifugation column.
  10. For mass measurements, take 1/3 of the solution (~66 µL) and split into three glass vials. Dry in a vacuum oven overnight at 40 °C under a vacuum of ~27 mmHg.
  11. Dissolve the other 2/3 of concentrated solution into 250 µL of PBS and store at 4 °C until further use.
  12. Prior to utilizing the FA-CuS NPs, sonicate them for 30 min in a bath sonicator on a high setting.

2. NP Characterization

  1. Perform dynamic light scattering (DLS) Add 10 µL of concentrated FA-CuS NPS in PBS solution from step 1.1.14 to 2 mL of DI water. Prior to characterization by DLS, sonicate the particles for 30 min in a bath sonicator on a high setting and filter through a 0.2 µm sterile filter to remove residual dust.
  2. Perform transmission electron microscopy (TEM) : Add 10 µL of concentrated FA-CuS NPS in PBS solution to a piece of wax paper. Invert a formvar coated copper grid on the top of the droplet and let sit for 2 min. Touch the edge of the formvar-coated grid to a piece of filter paper to remove excess liquid. Let air dry. Image the copper grid utilizing an electron microscope at an accelerating voltage of 80 kV.
    NOTE: Typical results from FA-CuS NPS characterization are presented in Figure 1.

3. Cell Culture

  1. This protocol utilizes SKOV-3 cells. Unless otherwise noted, culture SKOV-3 cells in McCoy's 5A medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin, and maintain at 37 °C in a humidified 5% CO2 incubator.

4. Fluorescent Tagging of FA-CuS NPS for Microscopy

  1. Add Texas-Red-X succinimidyl ester (0.2 mg dissolved in DMSO at a concentration of 10 mg/mL) to a solution containing 2 mg of FA-CuS NPs in 1 mL of 0.1 M NaHCO3 (pH ~9) buffer.
  2. Stir the reaction mixture using a magnetic stir bar for 1 h, away from light at room temperature.
  3. Concentrate the reaction mixture in a 4 mL 30 kDa MWCO centrifugation column by spinning at 4,000 x g for 10 min.
  4. Wash the concentrated solution 3x with 4 mL of 0.1 M NaHCO3 buffer (pH ~9) in a centrifugation column. Subsequently, wash the concentrated solution with 4 mL of DI water 3x or until only a trace amount of fluorescence remains visible in the flowthrough by UV-VIS.

5. FA-CuS NPS Uptake by Ovarian Cancer Cells

  1. Prior to incubation with FA-CuS NPs, incubate SKOV-3 cells in a T75 flask with 8−15 mL of folic-acid-free RPMI-1640 media with 10% FBS and 1% penicillin/streptomycin for at least 24 h.
  2. Seed cells in 0.5 mL of folic-acid-free RPMI-1640 complete growth media at a density of 0.1 x 106 cells/mL into a 24 well plate.
  3. The following day, incubate cells with 400 µg/mL FA-CuS NPS in 0.5 mL of folic-acid-free RPMI-1640 complete growth media for 2 h.
  4. Following this incubation, trypsinize the cells with 0.5 mL of 0.25% trypsin with EDTA. Add at least 1 mL of folic-acid-free RPMI-1640 complete growth media to neutralize the trypsin, and centrifuge the cells at 123 x g for 6 min.
  5. Remove the supernatant, resuspend the cells in 2 mL of PBS, and centrifuge at 123 x g for 6 min. Perform this wash step 2x to remove any unbound NPs.
  6. Resuspend the cells in 1−2 mL of PBS with 2% Tween solution.
  7. Count the cells using a hemocytometer and trypan blue. Further dilute cells if cell counts are too high. Dilute cells in PBS with 2% Tween to the chosen concentration for detection.
  8. The cells are now ready to be analyzed by the PAFC system.

6. Fluorescence Microscopy of FA-CuS NPS Uptake

  1. Repeat steps listed in step 5.1 and proceed with protocol below for microscopy.
  2. Seed cells at a density of 0.1 x 106 cells/mL in 0.5 mL of folic-acid-free RPMI-1640 complete growth media on glass coverslips in a 24 well plate.
  3. The following day, incubate the cells with fluorescently tagged FA-CuS NPs in triplicate, at concentrations of 100 µg/mL, 200 µg/mL, 300 µg/mL, and 400 µg/mL in 0.5 mL of folic-acid-free RPMI-1640 complete growth media.
  4. Incubate the cells with the NPs for 2 h in the 37 °C incubator
  5. Following this incubation period, wash the cells 3x with PBS.
    NOTE: For all wash steps, carefully add the solution on the side of the well plate to not disturb the cells. After addition, carefully tilt the plate and withdraw the solution from the side of the well.
  6. Incubate the cells with 0.5 mL of 3.7% paraformaldehyde (PFA) in PBS for 15 min and transfer the glass coverslips to a new 24 well plate.
    CAUTION: PFA is a known carcinogen. Do all fixation in a ventilated chemical fume hood and wear appropriate personal protective equipment.
  7. Incubate the cells in a solution of 3.7% PFA with 0.1% Triton-X in PBS for 5 min.
  8. Wash the cells with 0.5 mL of PBS 3x for 5 min each and transfer the coverslips to a new plate.
  9. Incubate the cells with 0.5 mL of a PBS solution containing DAPI (20 µL/mL of a 0.5 mg/mL stock solution is used for staining) for 5 min, away from light.
  10. Wash the cells with PBS 3x.
  11. Following the final PBS wash, mount the coverslips on slides with mounting medium.
  12. The cells are now ready to be imaged by fluorescence microscopy. Figure 2 shows an example of typical cell characterization by fluorescence microscopy.

7. Flow System Architecture

  1. Flow chamber construction
    NOTE:A SolidWorks file of a 3D printed flow tank can be found in the Supplementary Materials.
    1. Using the provided SolidWorks file, 3D print the flow tank using ABS thermoplastic or PLA plastic. Dimensions are provided below if SolidWorks is unavailable. Figure 3A shows a representation of this model. The body of the flow tank is 2.5 cm x 1.5 cm x 7.5 cm. The far ends of the flow tank include holes approximately 5 mm in diameter to allow for the entry of tubing containing the capillary tube.
      NOTE: The flow tank has a 1 cm hole, perpendicular to the orientation of the capillary tube, for placement of the ultrasound transducer. A cylindrical extrusion with the same inner diameter as the hole extends 6 mm into the tank. For real-time imaging, the flow tank has a 1 mm x 3 mm slot directly below the capillary tube.
    2. After printing the 3D flow tank, clean and assemble the system for use.
    3. Place glass coverslips over the 1 mm x 3 mm slot and the 1 cm hole in the flow system.
    4. Carefully seal with silicone to prevent leakage.
    5. Fit the capillary tube into the silicone cured tubes. Insert the tubes into the flow chamber though the side of the flow tank such that the glass capillary tube is directly above and in front of the 3 mm slot and the 1 cm hole.
    6. Seal the tubing using silicone to prevent leakage.
  2. Photoacoustic flow system setup
    NOTE: Figure 3B and Figure 3C show an example of the flow system architecture.
    1. Connect the transducer to an ultrasound pulser/receiver. Amplify the signal with a 59 dB gain.
    2. Connect the output of the filter to a multipurpose reconfigurable oscilloscope equipped with a built-in field programmable gate array.
    3. Connect one of the tubes coming from the flow chamber to a T-junction, connected to two syringe pumps at each branch.
    4. Fill one of the syringe pumps with air and the other pump with the sample to be analyzed. Set the pump containing air to a flow rate of 40 µL/min and the pump containing the sample to a flow rate of 20 µL/min. The resulting two-phase flow will produce sample volumes of 1 µL. At this flow rate, the system will test approximately 6.4 samples per minute.
      NOTE: To maintain a consistent distribution of cells, lightly vortex each sample immediately before being tested. In addition, rotate the syringe every few minutes in order to prevent the cells from settling in the solution.
    5. Connect the remaining tube exiting the flow system to a container with 10% bleach, to dispose of cells after they exit the flow system.
      NOTE: Before utilizing the flow system, check for leaks, as these can affect the flow. Cells must be contained within a closed system to maintain biological safety during the procedure.
    6. The design of the 3D printed tank allows for consistent and repeatable alignment between the transducer and laser light with minimal calibration. When placed correctly within the custom tank, the quartz capillary tube ensures that the transducer and laser are directly aligned.
    7. Place the section of the quartz capillary tube in direct alignment with the transducer, in the field of view of the microscope, allowing for careful placement of the optical fiber above the sample such that it illuminates the entire width of the tube.
    8. Irradiate the sample using an optical fiber channeling a diode-pumped solid state laser operating at a wavelength of 1,053 nm. The laser light incident on the sample and the transducer used to measure the photoacoustic effect are both unfocused.
    9. The energy of the laser incident on the sample is approximately 8 mJ and the 10 Hz laser rate is sufficient to illuminate each sample multiple times as it passes through the system.
    10. Place the flow system on top of an inverted microscope and ensure both the laser pulse and the path of the sample are visible as the sample passes though the flow system. Record flow using a microscope-mounted camera.
    11. Record the ultrasound acquisitions utilizing data acquisition software (see Table of Materials). Trigger ultrasound and pulsed laser using the FPGA. Utilize PBS with 2% Tween, and FA-CuS NPs at a concentration of 100 µg/mL in PBS 2% Tween, as negative and positive controls, respectively.
    12. Utilizing a microscope-mounted camera, record both the firing of the laser and the passage of samples though the flow system. These recordings will be utilized to correlate the acoustic signal recorded by the transducer with the firing of the laser. As the samples pass in front of the firing of the laser, the signal can then be correlated to the resulting photoacoustic signal for analysis. At a sampling rate of 10 Hz, the laser will illuminate each plug several times.

8. Post Processing

  1. For each signal acquisition, s(t), calculate the Hilbert transform, H[s(t)], in order to create an analytic signal.
  2. Create a complex envelope, se(t), by calculating the magnitude of the analytic signal, such that figure-protocol-13375 and integrate the envelope to measure the total signal resulting from each acquisition. Compare the signals from each test group (i.e., PBS, tagged cells, FA-CuS NPs, cells alone) utilizing a t-test in R statistical software. Raw photoacoustic signals and their Hilbert transforms are presented in Figure 4.
  3. For image reconstruction, normalize the complex envelope based on the maximum peak across the whole run. If comparing a series of runs, normalize the complex envelope using the maximum peak across the entire series. Following normalization, convert each acquisition into a series of pixel values. Represent each series of pixel values as a column in the image reconstruction. Representative reconstructions of PBS and the FA-CuS NPs signals are shown in Figure 5, where both images were normalized using the maximum peak across both runs.

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Results

Figure 1A shows a typical TEM image of the synthesized nanoparticles. The average size of the typical nanoparticle is approximately 8.6 nm ± 2.5 nm. Nanoparticle measuring was performed in ImageJ. Threshold and watershed functions were applied to separate the particles for measurement. The horizontal and vertical diameters of each particle were measured perpendicular to each other and further averaged. For DLS, a representative measureme...

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Discussion

This protocol is a straightforward method for the detection of ovarian CTCs utilizing PAFC and a targeted CuS contrast agent. Many methods have been explored for the detection of ovarian CTCs, including microfluidic devices, RT-PCR, and fluorescence flow cytometry23,24,25. These range in complexity, cost, and accuracy, limiting their effectiveness in clinical settings. PAFC introduces several advantages over these traditional me...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to acknowledge Madeleine Howell for her help with synthesis, Matthew Chest for his help designing the flow system, and Ethan Marschall for assistance with SolidWorks.

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Materials

NameCompanyCatalog NumberComments
0.025% Trypsin With EDTACorning25-053-Cl
0.2 µm 1,000 mL Vacuum Filtration UnitVWR10040-440For filtering larger volumes of DI water.
0.2 µm sterile syringe filterVWR28145-477
3D Printed TankCustom-made
Acquisition CardNational InstrumentsPXIe-5170R250 MS/s, 8-Channel, 14-bit
AlconoxSigma-Aldrich242985-1.8KGDetergent used for cleaning glassware.
Amicon Ultra-15 Centrifugal FiltersMilliporeUFC903024
Amicon Ultra-4 Centrifugal FiltersMilliporeUFC803024
Bright-Line HematocytometerHausser Scientific1492
Copper(II) ChlorideACROS ORGANICS206532500
Coupling ObjectiveThorlabsLMH-10x-532To couple pulsed light to optical fiber.
Coupling StageNewportF-91-C1-TStage for coupling pulsed light to objective. Holds FP-1A and LMH-10x-532
CPX Series Digital Ultrasonic Cleaning BathFisherbrandModel CPX3800
Data Acquisition softwareNational InstrumentsNI LabVIEW 2017 (32-bit)LabVIEW used to synchronize laser pulses with data acquisition.
Data Processing SoftwareMathworksMatlab R2016aReconstructions and graphs produced using Matlab software.
FBSSigma-AldrichF2442-500ML
Fiber ChuckNewportFPH-DJUsed to hold the bare fiber.
Fiber CouplerNewportFP-1A3-Axis stage for positioning fiber chuck and optical fiber at the focus of the objective.
Folic AcidSigma-AldrichF7876-10G
Formvar Coated TEM GridsElectron Microscopy SciencesFCF300-CU-SB
Masterflex TubingCole ParmerEW-96420-14
McCoy's 5A MediumATCC30-2007
Norm-Ject 10 mL SyringesHENKE SASS WOLF4100-X00V0
Optical FiberThorlabsFG550LECUsed to expose sample to pulsed light.
PBSAlfa AesarJ62036
Penicillin StreptomycinGIBCO15140-122
Pulsed LaserRPMC Lasers IncQuantus-Q1D-1053Pulsed laser source with specifications 1053 nm, 8 ns pulse, 10 Hz maximum.
Pulser/ReceiverOlympus5077PRReceives, filters, and amplifies photoacoustic signals. Operated with 59 dB Gain.
Quartz Capillary TubeSutter InstrumentQF150-75-10
RPMI Midum 1640 (1x) Folic Acid FreeGibco27016-021
SiliconeMomentive Performance Materials, Inc.GE284
SKOV-3 CellsATCCHTB-77
Sodium BicarbonateSigma-AldrichS5761
Sodium CarbonateSigma-AldrichS7795-500G
Sodium Hydroxide BeadsBDHBDH9292-500G
Sodium Sulfide NonahydrateSigma-Aldrich431648-50G
Syringe PumpsNew Era Pump Systems IncDUAL-1000
Texas Red-X-Succinimydl esterInvitrogen1949071
TransducerOlynmpusV214-BB-RMUltrasound detector with central frequency of 50 MHz and -6 dB fractional bandwidth of 82%.
Trypan Blue Solution 0.4%AmrescoK940-100ML
Tween 20Sigma-AldrichP7949-100ML
Ultrasound GelParker Laboratories Inc.Aquasonic 100Ultrasound gel for transducer coupling

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