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.
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.
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 cells3,4. 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 cancer5,6. 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-presentation11,12. EVs may provide immunostimulatory nucleic acids if they emerge under therapy-induced stress, facilitating an anti-tumor immune response13,14. Sensing of such RNA and DNA innate immune ligands in the tumor microenvironment has recently been shown to modulate responsiveness to checkpoint blockade significantly15,16,17. 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.
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
2. Immunization of mice with EVs
3. Flow cytometry analysis of splenic T cells
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 focuses on immunogenic cell death, cross-presentation, and EV-induced anti-tumor immunity, this protocol is restricted to the function of CD8+ cytotoxic T cells. As displayed in Figure 2, cells were gated as single cells, lymphocyte subset (by size and granularity), viable cells (excluding a life/dead marker), and CD3+ CD4– CD8+ cytotoxic T cells. Intracellular accumulation of IFN-γ was assessed as a surrogate marker for activation. Possible additional markers regarding T cell differentiation and exhaustion are discussed below.
Using the method described here, mice were immunized with EVs derived from OVA-expressing tumor cells cultured either under steady-state (untreated) or genotoxic stress conditions (oxaliplatin-treated). Only mice injected with EVs derived from tumor cells under genotoxic stress conditions induced potent activation of splenic cytotoxic T cells in recipient animals (Figure 3A). Injection of EVs derived from tumors under steady-state conditions resulted in some T cell activation, but that was not significantly different from T cell activation in mice injected with the PBS vehicle. These data show that under genotoxic stress, tumor cells can release potently immunogenic EVs. The production of IFN-γ was particularly increased when splenocytes of tumor EV-treated animals were ex vivo restimulated with the model tumor antigen OVA before analysis (Figure 3B). These data suggest that tumor-derived EVs can induce tumor antigen-specific immune responses. Interestingly, IFN-γ-production – even though to a much lesser extent – is also detected in the absence of antigen-specific restimulation. Possibly, other melanoma-associated antigens, such as the differentiation antigen TRP225, may be targeted by some part of the EV-induced T cell response.
Figure 1: Pictographic overview of the protocol. (A) Isolation procedure of EVs generated in tumor cell cultures resembling chemotherapy. (B) Schedule for the immunization of mice with EVs. (C) Staining protocol for flow cytometry analysis of cytotoxic T cells. Please click here to view a larger version of this figure.
Figure 2: Flow cytometry gating strategy to analyze cytotoxic T cell activation in the spleen. The numbers represent the percentage of its respective parent population. FSC-A: forward scatter area; FSC-H: forward scatter height; SSC: sideward scatter; live/dead: cell death marker. Please click here to view a larger version of this figure.
Figure 3: EVs derived from tumor cells under genotoxic stress can induce antigen-specific T cell responses in recipient animals. (A) Mice were immunized with EVs derived from tumor cells cultured either under steady-state (untreated) or genotoxic stress conditions (oxaliplatin-treated). Vehicle (PBS) injections were used as a negative control. IFN-γ production by cytotoxic T cells in the spleen upon EV immunization was determined. With this, splenic cell suspensions were restimulated with ovalbumin ex vivo before analysis. (B) Mice were treated with EVs derived from tumor cells under genotoxic stress conditions as described above. Splenic T cell activation was determined after ex vivo restimulation either in the presence or absence of ovalbumin. Bars depict the mean per group and whiskers its standard error. The one-way analysis of variance (ANOVA) test with Bonferroni posttest was used for multiple statistical comparisons of a dataset. The significance level was set at P < 0.05, P < 0.01, and P < 0.001 and is indicated here with asterisks (*, **, and ***). Please click here to view a larger version of this figure.
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 by screening through this protocol facilitates a more comprehensive understanding of conventional cancer therapies and enables a focused investigation into the role of EVs in cancer immunology.
Of note, experiments with EVs require some special considerations. In this protocol, EVs are semi-quantitatively normalized to the number of tumor cells released within 24 h. This approach reflects the aim to identify enhanced immunogenicity, regardless of whether it emerges from alterations in quality or quantity of released EVs. Therefore, reproducible in vivo results rely on the constant isolation efficacy of EVs. To this end, ensuring that EV pellets are resuspended entirely and promptly to avoid desiccation is a critical step.
Additionally, EVs must be quantified and characterized, e.g., by nanoparticle tracking analysis and western blot of canonical transmembrane, luminal, and at least one negative EV-marker20. Quantifying and characterizing EVs controls for inconstant isolation and addresses quantitative differences in EVs or EV subsets. This type of EV characterization constitutes a part of the minimum information that needs to be reported in studies about extracellular vesicles, according to the International Society of Extracellular Vesicles (ISEV) guidelines. However, various characterization methods are equally legitimate and should be selected concerning local availability and the individual research question. Notably, defining dosage of a substance of interest or ionizing irradiation constitutes another critical step of the protocol, which may require experimental validation to achieve an adequate level of cell death.
In general, potential contamination of isolated EVs with the treatment substance, soluble proteins, and lipoproteins must be considered. One strategy in this regard consists in reproducing the experiment with complementary EV-isolation techniques that the same type of contamination20 may not compromise. Immunoaffinity, for instance, isolates EVs with a lower yield but higher specificity than purely precipitation-based methods and may provide an appropriate control in this regard26. An alternative approach is to compare wild-type cell-derived EVs with isolations from genetically engineered cells with a specific deletion of genes involved in EV biogenesis or deploy substances that reduce EVs emission20.
Results obtained from this protocol should be complemented by a more comprehensive characterization of the EV-mediated immune response. Especially the classification of CD8+ T cells into effector and effector memory cells, through CD4427, as well as antigen-naïve and central memory cells, through CD62L28, may convey deeper insight. Furthermore, the analysis of T helper cells, regulatory T cells, and NK cells may be of interest. For testing the anti-tumor efficacy of EVs, mice may be challenged with the corresponding cancer cells after receiving EV immunization or treated with EVs against a preestablished cancer, thereby adapting the guidelines for detection of immunogenic cell death2,29 to this cell-free tumor derivate. However, conclusions from all these experimental setups are limited by the fact that cancer cells in a Petri dish potentially generate functionally different EVs than cancer cells embedded in a dynamic tumor microenvironment30 that often suppresses anti-cancer immunity. Thus, assessing EVs derived from tumor/fibroblast cocultures or ex vivo tumor tissue may better reflect the actual situation. In a next translational step toward clinical reality, EVs from patient material may be analyzed for immunogenicity before and during therapy to assess their usability as biomarkers.
As cancer-derived EVs were recently found to – under certain circumstances – modulate the immune system, the journey of exploring their clinical potential has only just begun31. For an in-depth analysis of the immune mechanisms co-opted by immunogenic EVs, useful tools comprise fluorescence microscopy visualizing the EV uptake by specific cells, the deployment of mice with genetic deficiencies for specific immune pathways, and screening methods for molecular alterations in the EV content. Ultimately, identifying immunogenic tumor-derived EVs, with screening methods described herein, will enable a better understanding of the underlying mechanisms behind EV-mediated immunity and therefore constitutes a crucial step toward harnessing their potential against cancer.
The authors have nothing to disclose.
This study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Projektnummer 360372040 – SFB 1335 and Projektnummer 395357507 – SFB 1371 (to H.P.), a Mechtild Harf Research Grant from the DKMS Foundation for Giving Life (to H.P.) a Young Investigator Award by the Melanoma Research Alliance (to S.H.), a scholarship by the Else-Kröner-Fresenius-Stiftung (to F.S.), a Seed Fund by the Technical University Munich (to S.H.) and a research grant by the Wilhelm Sander Foundation (2021.041.1, to S.H.). H. P. is supported by the EMBO Young Investigator Program.
AUTHOR CONTRIBUTIONS:
F.S., H.P., and S.H. designed the research, analyzed, and interpreted the results. F.S. and S.H. wrote the manuscript. H.P. and S.H. guided the study.
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) |