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

Summary

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Abstract

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

Introduction

The immune system plays a pivotal role in the fight against cancer, both when incited by immune checkpoint inhibition and for the efficacy of conventional cancer therapies. Tumor cells succumbing to genotoxic therapies such as the chemotherapeutic agents oxaliplatin and doxorubicin, or ionizing radiation treatment can release antigens and danger-associated molecular patterns (DAMPs) that potentially initiate an adaptive anti-tumor immune response¹. The most prominent DAMPs, in the context of immunogenic cell death, include find-me signals such as chemotactic ATP, eat-me signals such as the exposure of calreticulin, that promotes tumor cell uptake by antigen-presenting cells, and the release of HMGB1, that activates pattern recognition receptors, thereby enhancing the cross-presentation of tumor antigens². Furthermore, type I interferons (IFN-I), induced via tumor-derived immunogenic nucleic acids or other stimuli, are sensed by dendritic cells, enabling them to effectively prime tumor-specific cytotoxic T cells^3,4. Clinically, activated and proliferating CD8⁺ T cells infiltrating the tumor provide an independent prognostic factor for prolonged survival in many cancer patients. Released from such activated T cells, IFN-γ mediates direct antiproliferative effects on cancer cells and drives Th1 polarization and cytotoxic T cell differentiation, thereby contributing to effective immunosurveillance against cancer^5,6. Oxaliplatin is a bona fide immunogenic cell death inducer, mediating such adaptive immune response against cancer⁷. However, the plethora of initial immunogenic signals released by tumor cells under therapy-induced stress remains to be fully unveiled. Despite significant advances in cancer immunotherapy, expanding its benefits to a larger portion of patients remains a challenge. A more detailed understanding of immunogenic signals that initiate T cell activation may guide the development of novel therapies.

A heterogeneous group of membrane-enclosed structures, known as extracellular vesicles (EVs), seem to serve as intercellular communication devices. Emitted by virtually all cell types, EVs carry functional proteins, RNA, DNA, and other molecules to a recipient cell or may alter a cell's functional state just by binding to receptors on the cell surface. Their biologically active cargo varies significantly by the type and functional state of the generating cell⁸. In cancer immunology, EVs released from tumor cells have been predominantly regarded as adversarial to immunotherapy because they eventually promote invasive growth, preform metastatic niches⁹, and suppress the immune response¹⁰. In contrast, some studies have shown that EVs can transfer tumor antigens to dendritic cells for effective cross-presentation^11,12. EVs may provide immunostimulatory nucleic acids if they emerge under therapy-induced stress, facilitating an anti-tumor immune response^13,14. Sensing of such RNA and DNA innate immune ligands in the tumor microenvironment has recently been shown to modulate responsiveness to checkpoint blockade significantly^15,16,17. Hence, the immunogenic role of EVs released by tumor cells under different therapy-induced stress needs to be further elucidated. Since EVs constitute a young yet growing field of research, standardization of methods is still ongoing. Therefore, sharing knowledge is essential to improve the research reproducibility on interactions between EVs and cancer immunology. With this in mind, this manuscript describes a simple protocol to assess the immunogenic effect of tumor-derived EVs in vivo.

This assessment is performed by generating tumor-derived EVs, immunizing recipient mice with those EVs, and analyzing splenic T cells via flow cytometry. EV generation is ideally performed by seeding murine tumor cells in an EV-free cell culture medium for a high degree of purity. Cells are treated with a specific cell stress stimulus, such as chemotherapy, to compare the effect of therapy-induced EVs against baseline immunogenicity of respective tumor-derived EVs. The isolation of EVs may well be performed by various techniques that should be selected according to in vivo applicability and local availability. The following protocol describes a precipitation-based assay with a commercial kit for EV purification. Mice are immunized twice with those EVs. Fourteen days after the first injection, T cells are extracted from the spleen and analyzed for IFN-γ production via flow cytometry to evaluate a systemic immune response. With this, the potential of tumor-derived EVs, emerging under different therapeutic regimens, to induce anti-tumor T cell responses is assessed relatively easily, quickly, and with high validity¹³. Therefore, this method is suitable for an immunological screening of EVs derived from cancer cells under various conditions.

Protocol

At the onset of experiments, mice were at least 6 weeks of age and were maintained under specific pathogen-free conditions. The present protocol complies with the Institutional ethical standards and prevailing local regulations. Animal studies were approved by the local regulatory agency (Regierung von Oberbayern, Munich, Germany). Possible sex-related biases were not investigated in these studies.

1. Generation and isolation of EVs derived from tumor cells after chemotherapy exposure

1. Culture murine B16 melanoma cells expressing ovalbumin (B16-OVA) in DMEM (containing 4 mM L-glutamine and 4.5 g/L D-glucose) supplemented with FCS (10% v/v), penicillin (100 Units/mL), and streptomycin (100 μg/mL) at 37 °C, until cells grow steadily and are approximately 90% confluent.
   NOTE: Perform this section of the protocol entirely under sterile conditions using a cell culture hood. For analysis of other cancer entities, cell lines expressing a potent antigen are preferable to assess antigen-specific T cell responses, as they allow for specific ex vivo antigen restimulation.
2. To prepare the cell culture media required for EV generation, deplete bovine EVs within FCS by ultracentrifugation at 100,000 x g for 24 h at 4 °C, and then discard the pellet. Alternatively, choose a commercial preparation low in animal EVs beforehand.
3. Harvest B16-OVA cells and wash them twice in PBS, and then seed at a concentration of 400,000 cells/mL in EV-depleted media.
   NOTE: Adapt the cell concentration to the growth dynamic of the cell line under investigation so that cells do not overgrow.
4. Treat B16-OVA cells by adding 30 μg of oxaliplatin per mL and incubate for 24 h at 37 °C. Leave control conditions untreated. At the first use of a genotoxic substance, titrate the desired cytotoxic efficacy using cell-viability assays such as trypan blue exclusion¹⁸.
   NOTE: This assay may also evaluate EVs generated in cell cultures treated with other immune-modulating substances or ionizing irradiation besides chemotherapeutics.
   CAUTION: Oxaliplatin causes skin and severe eye irritation and may cause an allergic skin reaction and respiratory irritation. Oxaliplatin is suspected of causing cancer. As a precaution, use personal protective equipment, including adequate gloves, goggles, masks, and clothing, cleaned before reuse. Avoid inhalation and wash hands thoroughly after handling. Avoid release into the environment and dispose oxaliplatin according to prevailing regulations. Obtain detailed information from the safety data sheet.
5. Collect cell culture supernatant. Centrifuge first at 400 x g for 5 min at 4 °C, and then at 2,000 x g for 30 min at 4 °C, each time discarding the pellet. Finally, filter through a 220 nm PVDF membrane. Use a fresh tube for each step to remove any cell debris.
   NOTE: At this stage, the EV-containing supernatant may be stored at 4 °C for a day before resuming the protocol. However, it is strongly recommended to adhere to the described schedule with immediate EV purification.
6. Mix 1 mL of supernatant with 0.5 mL of a specific commercially available exosome isolation reagent (see Table of Materials) in a V-shaped 1.5 mL tube. Thoroughly pipette up and down or vortex to create a homogenous solution. Incubate overnight at 4 °C.
7. Centrifuge at 10,000 x g for 60 min at 4 °C. Carefully discard the supernatant. Remove the remaining drops by tapping the 1.5 mL tube upside down on a paper towel and by aspiration through a pipette with a fine tip without touching the EV-pellet at the bottom.
   1. Thoroughly remove all fluids to prevent uncontrolled dilution of the EV-pellet. Also, execute these tasks quickly to prevent the pellet from drying out.
8. Resuspend the EVs in cold PBS by pipetting up and down without scratching the pellet from the tube's wall with the tip. Now, transfer the suspension step by step from the first to the last tube to pool the EVs.
   NOTE: Use a volume of PBS that equals 5 μL multiplied by the number of tubes. 5 μL of the final suspension contains the isolated EVs released from 400,000 cells under chemotherapy or at a steady state.
9. Preferably, use EVs directly. If this is not possible, store EV suspensions at -80 °C in siliconized vessels for up to 28 days until application.
   NOTE: The EVs described here and in several other publications do not lose their respective biological function when stored at -80 °C for that time period¹⁹.
10. Quantify and characterize EV isolates according to the MISEV2018 guidelines²⁰.
    NOTE: Possible methods for quantification include nanoparticle tracking analysis (NTA)²¹ and the detection of EV's membrane-bound proteins²². Possible approaches to further characterize EVs include electron microscopy²³ and western blot²⁰.

2. Immunization of mice with EVs

1. Plan the in vivo experiment with C57BL/6 mice (or other syngeneic mice corresponding to the tumor cell line), including treatment groups receiving EVs derived from treated cells, untreated cells, and PBS (vehicle), respectively.
   NOTE: Preferably, use mice at the age of 6-8 weeks to prevent physiological senescence from diminishing the immune response²⁴.
2. Mix 5 μL of EV-suspension with 55 μL cold PBS for each mouse within the respective treatment group to immunize it with EVs isolated from 4.0 x 10⁵ B16-OVA cells.
   NOTE: This amount of EVs corresponds to approximately 2 x 10⁹ particles measured by nanoparticle tracking analysis (data not shown). OVA protein mixed with an adjuvant (e.g., LPS) can be applied as a potent vaccine positive control.
   1. Fill syringes (needle size 26-30 G) with 60 μL of the diluted EVs or PBS, respectively and put immediately on ice.
      NOTE: In the protocol, the amount of injected EVs is normalized to the number of EV-releasing tumor cells to experimentally consider both qualitative and quantitative effects of oxaliplatin on tumor cell EV biogenesis. For some readers, normalization to a specific concentration of produced EVs may better fit their experimental setup depending on their scientific question.
3. Inoculate EVs or PBS subcutaneously into the medial aspect of the mice's thigh and repeat the immunization after 7 days. Fourteen days after the first treatment, sacrifice mice, e.g., by cervical dislocation to analyze the immune response.
   ​NOTE: Alternative subcutaneous injection routes may be used, according to the local standards.

3. Flow cytometry analysis of splenic T cells

1. Prepare and cool complete RPMI (cRPMI), supplementing RPMI-1640 with FCS (10% v/v), penicillin (100 Units/mL), streptomycin (100 μg/mL), L-glutamine (2 mM), and β-mercaptoethanol (50 μM).
2. Resect the spleen from the opened abdominal cavity. Mash the spleen with a moistened 100 μm cell strainer and the plastic plunger of a syringe and flush the splenic cells into a 50 mL tube with 5-10 mL of cRPMI. Centrifuge at 400 x g for 5 min at 4 °C and discard the supernatant.
   NOTE: Keep cells on ice whenever possible. To analyze the local rather than the splenic immune response, resect the draining popliteal and inguinal lymph nodes, following the same protocol. In this case, skip the next step for the lysis of erythrocytes.
3. To remove erythrocytes from the cell suspension, resuspend the pellet with 2 mL of red blood cell lysis buffer (see Table of Materials) and incubate for 5 min at room temperature. Then, stop the reaction by adding cRPMI. Centrifuge at 400 x g for 5 min at 4 °C and discard the supernatant.
4. For seeding, resuspend the cell pellet in cRPMI and count the cells to place triplicates of 200,000 cells with 200 μL cRPMI into each well, using a 96-well plate with a U-shaped bottom. Incubate for 48 h at 37 °C.
   NOTE: To address the antigen-specificity of activated T cells, add 1 μg/mL soluble ovalbumin (or another tumor antigen corresponding to cell line under investigation) or leave without additional stimulus, respectively. Adding the immune-dominant peptide epitope SIINFEKL, instead of full-length ovalbumin, allows for a shorter incubation period. Besides flow cytometry, the mice's serum and the cell culture supernatant after 48 h of incubation can be analyzed for various cytokines.
5. After 48 h, to enhance intracellular IFN-γ staining by, inter alia, blocking the Golgi-mediated secretion of proteins, add Brefeldin A (5 ng/mL), PMA (20 ng/mL), and Ionomycin (1 µg/mL) to the cell culture. Incubate for 4 h at 37 °C.
6. Before staining surface biomarkers, transfer splenocytes to a 96-well plate with a V-shaped bottom and wash twice with PBS. Then, add fluorescent antibodies, compatible with the locally available flow cytometer, directed against surface biomarkers, CD3, CD8, and CD4 (see Table of Materials), diluted 1:400, plus a fixable viability dye, diluted 1:1,000 in PBS. Resuspend pelleted splenocytes in the staining solution and incubate for 30 min at 4 °C, protected from light.
7. For fixation and permeabilization of splenocytes, wash twice in FACS-buffer (PBS plus 3% v/v FCS), and then resuspend in 100 µL of fixation/permeabilization buffer (see Table of Materials) per well. Incubate for 30 min at 4 °C, protected from light.
8. For staining of intracellular IFN-γ, wash splenocytes in fixation/permeabilization buffer and resuspend with fluorescent antibodies against IFN-γ, diluted 1:200 in the buffer. Incubate for at least 1 h (up to a maximum of 12 h) at 4 °C, protected from light.
9. Before measuring the samples by flow cytometry, wash splenocytes twice in fixation/permeabilization buffer and resuspend in FACS-buffer. Analyze the activation of cytotoxic T cells according to the gating strategy displayed in Figure 2.
   1. First, to detect single cells, blot FSC-H against FSC-A. Then, to detect lymphoid cells, blot SSC against FSC-A. Subsequently, select living CD3⁺, CD4^-, CD8⁺ cells and determine their IFN-γ-producing subset to quantify the activation of cytotoxic T cells in the spleen.
      NOTE: Include a Fluorescence-minus-one (FMO) stain with all fluorochromes except the fluorochrome targeted against IFN-γ as negative technical control.

Results

This protocol is intended to facilitate the straightforward and easily reproducible assessment of the immunogenicity of tumor-derived EVs. Hereby, mice are inoculated with EVs derived from in vitro cultures of tumor cells expressing the model antigen chicken ovalbumin (OVA). The subsequent immune response is analyzed in splenic T cells via flow cytometry.

Figure 1 gives an overview of the practical steps of the entire protocol. Since the work foc...

Discussion

This protocol provides an immunological in vivo assessment of EVs derived from melanoma cells under chemotherapy-induced stress while adapting to EVs emitted from various cancers under various treatments. Immunizing mice with EVs derived from oxaliplatin-treated B16-OVA cells, for instance, expands IFN-γ-producing CD8⁺ T cells in the spleen, which are further stimulated by ex vivo incubation with ovalbumin, indicating a tumor-specific immune response. Thus, detection of immunogenic EVs b...

Materials

Name	Company	Catalog Number	Comments
Anti-CD3 FITC	Biolegend	100204	Clone 17A2
Anti-CD4 PacBlue	Biolegend	100428	Clone GK1.5
Anti-CD8 APC	Biolegend	100712	Clone 53-6.7
Anti-IFNγ PE	eBioscience	RM90022	Clone XMG1.2
Brefeldin A	Biolegend	420601	Brefeldin A Solution (1,000x)
Cell Strainer, 100 µm	Greiner	542000	EASYstrainer 100 µm
DMEM	Sigma-Aldrich	D6429	Dulbecco's Modified Eagle's Medium with D-glucose (4.5 g/L) and L-glutamine (4 mM)
FBS Good Forte	PAN BIOTECH	P40-47500	Fetal Calf Serum (FCS)
Fixable Viability Dye eFluor 506	eBioscience, division of Thermo Fischer Scientific	65-0866-14
Fixation/Permeabilization Concentrate	eBioscience	00-5123-43	Fixation/Permeabilization Concentrate (10x)
Fixation/Permeabilization Diluent	eBioscience	00-5223-56
Ionomycin	Sigma-Aldrich	407952	From Streptomyces conglobatus - CAS 56092-82-1, ≥ 97% (HPLC)
L-Glutamine	Gibco	25030-032	L-Glutamine (200 mM)
Ovalbumin	InvivoGen	vac-pova	Ovalbumine with < 1 EU/mg endotoxin - CAS 9006-59-1
Oxaliplatin	Pharmacy of MRI hospital
PBS	Sigma-Aldrich	D8537	Phosphate Buffered Saline without calcium chloride and magnesium chloride
Penicillin-Streptomycin	Gibco	1514-122	Mixture of penicillin (10,000 U/mL) and streptomycin (10,000 ug/mL)
PMA	Sigma-Aldrich	P1585	Phorbol 12-myristate 13-acetate, ≥ 99% (HPLC)
PVDF filter, 0,22 µm, for syringes	Merck Millipore	SLGV033RS	Millex-GV Filter Unit 0.22 µm Durapore PVDF Membrane
Red Blood Cell Lysis Buffer	Invitrogen	00-4333-57
RPMI 1640	Thermo Fischer Scientific	11875	Roswell Park Memorial Institute 1640 Medium with D-glucose (2.00 g/L) and L-glutamine (300 mg/L), without HEPES
Syringe, 26 G	BD Biosciences	305501	1 mL Sub-Q Syringes with needle (0.45 mm x 12.7 mm)
Total Exosome Isolation Reagent	Invitrogen	4478359	For isolation from cell culture media
β-Mercaptoethanol	Thermo Fischer Scientific	31350	β-Mercaptoethanol (50 mM)