In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This video shows how to assess the in vivo immunogenicity of tumor-derived extracellular vesicles, or EVs, using flow cytometry. The screening method described herein is relatively easy to perform and can be adapted to various settings. In cancer, identifying conditions of immunogenic extracellular vesicle releases of particular translational relevance.

As such, autologous EVs might be used as personalized anti-cancer treatments. Demonstrating the first part of the procedure will be Tatiana Nedelko, a technical assistant from my laboratory. To start the preparation of cell culture media required for extracellular vesicle, or EVs, deplete the bovine EVs in FCS media by ultracentrifugation at 100, 000 times g for 24 hours at 4 degrees Celsius.

Discard the pellet after centrifugation. Harvest the murine B16 melanoma cells expressing ovalbumin, or B16-OVA cells, from the culture and wash the cells twice with PBS. Then, seed the cells at a concentration of 400, 000 cells per milliliter in the EV-depleted media.

Next, treat the B16-OVA cells with 30 micrograms of oxaliplatin per milliliter and incubate for 24 hours at 37 degrees Celsius with untreated control samples. After 24 hours, collect the supernatant to centrifuge at 400 times g for five minutes at 4 degree Celsius. Discard the pellet before centrifugation again at 2, 000 times g for 30 minutes at 4 degrees Celsius.

After discarding the pellet, filter the supernatant through a 220 nanometer PVDF membrane filter into a fresh tube. Then, mix 1 milliliter of supernatant with 0.5 milliliters of a specific commercially-available exosome isolation reagent. Vortex and transfer the mixture into 1.5 milliliter tubes, followed by overnight incubation at 4 degrees Celsius.

The next day, centrifuge the mixture at 10, 000 times g for 60 minutes at 4 degrees Celsius. After carefully discarding the supernatant, remove the remaining drops by tapping the 1.5 milliliter tube upside down on a paper towel and by aspiration through a fine tip of pipette without touching the EV pellet at the bottom. Resuspend the pellet in cold PBS by pipetting up and down without scratching the pellet with the tip.

Then, pool the EVs from all tubes. If not using immediately, store the EV suspensions at 80 degrees Celsius in siliconized vessels for up to 28 days until application. To immunize each mouse in the treatment group with EVs isolated from 4.0 x 10 to the fifth B16-OVA cells, mix 5 microliters of the EV suspension with 55 microliters of cold PBS.

Next, fill syringes mounted with 26 to 30-gauge needles with 60 microliters of the diluted EVs or PBS and then immediately put the syringe on ice. Inoculate the EVs or PBS subcutaneously into the medial aspect of the mice's thigh. Repeat the immunization after seven days.

14 days after the first treatment, sacrifice the mice to resect the spleen. Then, mash the resected spleen with a moistened 100-micrometer cell strainer and the plastic plunger of a syringe. Flush the splenic cells into a 50 milliliter tube with 5 to 10 milliliters of the cool complete RPMI.

Next, centrifuge the cells at 400 times g for five minutes at 4 degrees Celsius before discarding the supernatant. To remove erythrocytes from the cell suspension, resuspend and incubate the cell pellet with 2 milliliters of the red blood cell lysis buffer for five minutes at room temperature. Then, stop the reaction by adding five to 10 milliliters of complete RPMI, followed by centrifugation as described earlier.

After resuspending the cell pellet in complete RPMI, count the cells from every mouse and place the triplicates of 200, 000 splenocytes with 200 microliters of cRPMI into each well of a 96-well plate with a U-shaped bottom. Then, incubate the plates for 48 hours at 37 degrees Celsius. After 48 hours, treat the cell culture with 5 nanograms per milliliter Brefeldin A, 20 nanograms per milliliter PMA, and 1 microgram per milliliter Ionomycin at 37 degrees Celsius for 4 hours.

Post incubation, transfer the splenocytes to a 96-well plate with a V-shaped bottom and wash the cells twice with PBS. Resuspend the pelleted splenocytes in the freshly-prepared staining solution and incubate the cell suspension for 30 minutes at 4 degrees Celsius protected from light. For fixation and permeabilization, wash the splenocytes twice in the FACS-buffer and then resuspend the splenocytes in 100 microliters per well of the fixation and permeabilization solution, followed by incubation in the dark as explained earlier.

For staining the intracellular interferon gamma, wash the splenocytes in the fixation or permeabilization buffer before resending the cells with the fluorescent antibodies against interferon gamma diluted 1:200 in the buffer. Incubate the splenocytes for 1 to 12 hours at 4 degree Celsius in the dark. After thorough washing and resuspension in FACS-buffer, analyze the activation of cytotoxic T cells according to the gating strategy explained in the manuscript.

In the representative image, the gating strategy to analyze the activation of cytotoxic T-cells is displayed. The cells were gated as the single cells, the lymphocyte subset, the viable CD3-positive T cells, and the CD4-negative CD8-positive cytotoxic T-cells. The intracellular accumulation of the interferon gamma was assessed as a surrogate marker for the activation.

After the immunization with EVs derived from the tumor cells, the induction of antigen-specific T cell responses in recipient mice was studied. The tumor-derived EVs cultured under genotoxic stress conditions, such as oxaliplatin treatment, induced potent activation of the splenic cytotoxic T cells in contrast to untreated EVs. The vehicle-treated group of animals showed the lowest response.

The activation of splenic T cells was determined after the ex vivo restimulation either in the presence or absence of ovalbumin. The production of the interferon gamma was increased when the splenocytes of tumor EV-treated animals were restimulated with the model tumor antigen ovalbumin before the analysis. Please note that reproducible results rely on the constant isolation efficacy of EVs.

Thus, ensure that EV pellets are resuspended entirely and promptly to avoid desiccation. With this protocol, we were able to analyze specific pathways within tumor cells that determine the immunogenicity of tumor cell-derived EVs. This may pave the road to harness such immunogenic EVs in cancer treatment.