Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Nanoplasmon-enhanced scattering, or nPES, is a simple and noninvasive alternative to the surgical biopsy that can skip time-consuming and labor-intensive exosome purification steps, expanding the application of exosome analysis to clinical settings. This nPES assay is a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes that does not require separate isolation and purification steps. We only need a very small clinical sample for this assay.

Therefore, this method can expand the use of nPES to studying genetically engineered or PDX mouse models. There are a few steps in this protocol that might seem daunting. However, with a few practice runs, everyone with basic wet lab skills can learn to successfully perform nPES.

To begin the procedure, combine 40 microliters of a suspension of NeutrAvidin-functionalized gold nanorods with 200 microliters of cold, pH seven phosphate-buffered saline. Centrifuge the mixture at 8500 times g at four degrees Celsius for 10 minutes, and remove the supernatant. Repeat this washing process twice more, and then resuspend the nanorods in 40 microliters of cold PBS.

Next, add 150 microliters of cold PBS and 10 microliters of a 0.5-milligram-per-milliliter solution of the appropriate biotinylated antibodies to the suspension. Mix at four degrees Celsius for two hours to obtain antibody-conjugated gold nanorods. Wash the nanorods three times by centrifugation in 200-microliter portions of cold PBS at 6500 times g at four degrees Celsius for 10 minutes each.

Afterwards, resuspend the washed antibody-conjugated nanorods in 200 microliters of cold PBS, and store them at four degrees Celsius for up to 24 hours. To begin preparing the slide, dilute the desired exosome capture antibodies to 025 milligrams per milliliter in PBS. Pipette one microliter of this solution into each well of a protein A/G-treated slide backed with optical-grade glass.

Then, transfer the slide to a humidifying box to ensure wells do not dry up during incubation. Incubate the slide at 37 degrees Celsius for one hour to immobilize the capture antibodies. Then, aspirate the remaining solution to remove unbound antibodies.

Wash the wells by adding and aspirating one microliter of PBS three times. Next, quickly load each well with one microliter of PBS-based blocking buffer, and incubate the slide at 37 degrees Celsius for two hours. When running around 15 samples, we must use a single-channel pipette to load blocking buffer onto 120 wells in less than five minutes to avoid the evaporation of blocking agent.

Start preparing a solution of antibody-conjugated gold nanorods during slide blocking. About 30 minutes before blocking finishes, rapidly thaw plasma or serum samples in a room-temperature water bath. Vortex the thawed samples for 30 seconds to ensure that the suspensions are homogeneous.

Then, centrifuge the samples at 500 times g for 15 minutes to precipitate protein aggregates and other debris. Transfer 10-microliter aliquots of the supernatant to fresh tubes, and make one-to-one dilutions with PBS. Mix the diluted samples by gentle vortexing or inversion as appropriate.

When slide blocking has finished, aspirate the blocking buffer and wash the wells three times with one-microliter portions of PBS. Immediately load the samples into the wells with one microliter per well and eight replicates per sample. Load exosome standards into the appropriate wells in the same way.

Incubate the slide for 12 to 18 hours in a refrigerator at four degrees Celsius. Then, aspirate the wells, and wash each well once with one microliter of PBS. Load one microliter of the antibody-conjugated gold nanorod suspension into each well, and incubate the slide at 37 degrees Celsius for two hours.

Afterwards, aspirate the nanorod suspension, and wash the slide in PBS supplemented with 1%polysorbate 20 for 10 minutes using a mixer. Then, aspirate the wells, and wash the slide in deionized water for 10 minutes on a rotating mixer. Remove the water, and allow the slide to air-dry in a clean Petri dish before bringing the slide to the dark-field microscope.

Set up an inverted microscope equipped with a dark-field oil-immersion condenser, a 4x objective, and a motorized stage. Connect the microscope to a computer via a digital camera, and open the imaging software. Then, place the sample slide upside down on the microscope stage, and apply a small drop of immersion oil where the condenser lens contacts the slide.

Display the view through the microscope in the imaging software. Adjust the exposure time against a high-concentration standard well to ensure that the image will not be saturated. Then, open the tool for scanning large images, and set the objective magnification to 10x.

Choose to close the active shutter during stage movement and to wait 20 milliseconds before each capture. Set the stitching overlap to 20%and select stitching via the optimal path. Choose to create a large image from the scan.

Set the camera to focus manually at the start of the scan, and enable automatic step-by-step focus every 20 fields. Then, choose to set left, right, top, and bottom limits for the target scan field, and define the scan area by moving the microscope stage to each of the desired limits. Next, adjust the focus, the condenser, and the area lighting to obtain a clear, well-lit image.

Name the image file to be created, and start the scan. When it finishes, open the image and save it at 1/8 scale. Open the image analysis software.

Then, open the Plugins menu, click DSM Scan, define the number of columns and rows, set resize percentage to 25, set spot diameter, in pixels, to 190 to 200, set diameter range to 32, and set increment diameter, in pixels, to eight. Set the low and high DSM limits to zero and 62, the adjacent distance to 100, and the subtraction bias to zero. Run the DSM algorithm, and save the resulting data.

The DSM analysis technique showed good reproducibility across serially diluted PANC-1 exosome samples ranging from 0.24 to 1.2 micrograms per microliter. A strong linear correlation was observed between the scatter response from the gold nanorods and the exosome protein concentration. A significant difference in abundance of serum exosomes expressing the cancer-associated biomarker EphA2 was observed between patients with pancreatic cancer and healthy patients.

From the addition of blocking agent onward, fast and accurate pipetting is crucial to avoid the evaporation of samples, introducing cross-contamination, and scratching the surface of the slide. Following this procedure, we perform statistic analysis to investigate any correlation between the expression of the exosomal biomarker of interest and different stage of the disease that is being studied. After establishing this technology, and we're able to find exosomal biomarkers for early-stage pancreatic cancers.

Currently, we're expanding this discovery to other types of cancers and infection disease. Usually, the samples handled in this assay are either patient samples or purified exosome samples from cancer cell lines, which necessitate special safety training.