Table 1.

A summary of vaccine studies utilizing tumor-derived exosomes in in vitro and in vivo preclinical animal models.

Source of tumor exosomes	Purification method	Aim of study	Treatment regimen	Key results	Ref.
-HeLa cell line	Ultracentrifugation/sucrose gradient	In vitro investigation of HeLa-derived exosomes immunogenicity.	Capacity of HeLa-TEX for inducing immune responses was examined.	Loading with HeLa‑TEX-induced DC maturation, in vitro T lymphocyte proliferation and specific CTL activity.	135
-EG7 expressing ovalbumin (OVA- transfected EL4 mouse thymoma) cell line	Ultracentrifugation	Comparison between TEX and dendritic cell-derived exosomes (DEXs) based vaccination.	Melanoma-bearing mice were immunized intravenously with OVA pulsed DC-derived exosomes (EXODC) or tumor-derived exosomes (TEXEG7) (10 µg/mouse).	EXODC were more immunogenic compared to TEXEG7. EXODC more strongly stimulated CD8+T cell proliferation and differentiation in compared to TEXEG7. EXODC induced more efficient CTL activity and stronger antitumor immunity both in vitro and in vivo compared to TEXEG7.	11
-EG7 expressing ovalbumin (OVA- transfected EL4 mouse thymoma) cell line	Ultracentrifugation	Comparison of immunogenicity between OVA protein pulsed DC-derived exosomes (EXODC) and DC pulsed with TEXOVA (DCEXO).	Melanoma-bearing mice were treated intravenously with EXODC or DCEXO vaccines.	TEX uptake by DC was mediated by LFA-1, CD54, and CLR. DCEXO stimulated OVA-specific T cell proliferation in vitro and in vivo. DCEXO more strongly eradicated established tumors and induced protection against lung metastasis.	144
-A20 (H-2d) mouse B lymphoma/leukemia cell line (Exo) -Heat-shocked A20 (H-2d) mouse B lymphoma/leukemia cell line (HS-Exo) - murine colon cancer CT-26 cell line	Ultracentrifugation	Evaluation of heat shock induction in improvement of exosome-based tumor vaccines.	Lymphoma/leukemia-bearing BALB/c mice were immunized subcutaneously with TEX and HS-TEX (10 µg/mouse)	Heat-shock treatment did not affect the amount of A20 exosomal protein secretory rate, in contrast increased the exosomal proteins relevant to efficient immune responses. HS-TEX contained more MHCI, HSP60, HSP90, RANTES, and IL-1b compared to TEX and CD40 was only in HS-TEX. Efficacy of both therapeutic and prophylactic vaccines was investigated. 80% of the vaccinated mice with HS-TEX remained tumor-free 90 days after tumor challenge and showed higher levels of IFN-γ and IL-2 compared with TEX-immunized mice (p < .05).	28
- B16F1 murine melanoma cell line - CIITA gene (MHC class II transcriptional activator) transduced B16F1cell line	Ultracentrifugation/sucrose gradient	Investigation of CIITA transduction effect in enhancing immune stimulation as compared to exosomes from parental tumor cells (TEX).	-For preventive model, C57BL/6 mice were intradermally vaccinated with PBS, TEX or CIITA-TEX (5 or 20 µg/mouse), and then challenged subcutaneously with melanoma B16F1 cells. -For therapeutic model, B16F1 melanome-bearing mice were immunized intradermally with PBS, TEX and CIITA-TEX (20 µg/mouse).	MHC class II molecules were highly enriched in CIITA-TEX as compared to parental TEX. It was found that in vitro loading of DCs with CIITA-TEX significantly induced higher level expression of immunostimulatory molecules such as MHC class II molecules and CD86 compared with DCs loaded with TEX. Increased proliferation and IL-2 production of naïve splenocytes were observed in mice treated with CIITA-TEX vaccine when compared to mice treated with parental TEX. Mice treated with CIITA-TEX had improved preventive and therapeutic anti-tumor immune response by both cellular and humoral mechanisms in a dose-dependent manner with increased survival rate and the best control of tumor growth.	29
-Murine colon cancer CT-26 cell line -CIITA gene transduced CT-26 cell line	Ultracentrifugation/sucrose gradient	Transduction of CIITA gene into CT26 cell line and evaluation of immune response induction by CT-26-CIITA TEX compared to parental CT-26 TEX.	Balb/c mice were intradermally immunized with PBS, CT-26 TEX and CT-26 -CIITA TEX (10 or 20 µg/mouse), and challenged subcutaneously with CT-26 cells.	CT-26 -CIITA TEX expressed high level of MHC class II molecules and promoted DC maturation. Preventive CT-26-CIITA TEX vaccinated mice models showed high expression of TNF-α, IFN-γ, IL-12 cytokines, and decrease in IL-10 expression and also effectively inhibited tumor growth in compared to CT-26 TEX.	30
-K562 leukemia cell line -L1210 acute lymphoblastic leukemia cell line	Ultracentrifugation/sucrose gradient	Examination of L1210-TEX and DC- L1210 TEX ability in induction of protective antitumor immunity in leukemia mice model.	- For therapeutic model, DBA/2 mice were immunized subcutaneously with PBS, L1210-TEX (30 µg/mouse), non-pulsed DC and DC-L1210 TEX (1.0–4.0 × 106cells/mouse), then challenged with L1210 leukemia cells. - For preventive model, after challenging with L1210 leukemia cells, DBA/2 mice were immunized.	L1210-TEX harbored ABL specific protein from their parental cells as well as HSP70 that facilitates antigen presentation. Both L1210-TEX and TEX-pulsed DCs induced antigen-specific anti-leukemic CTL immune response ex vivo and in vivo. L1210 TEX-pulsed DCs were more potent in protective and therapeutic anti-leukemic immunity induction compared to L1210-TEX alone.	31
-Myeloid leukemia WEHI3B cell line -Renal cell carcinoma (RENCA) cell line	Ultracentrifugation/sucrose gradient	Efficacy evaluation of TEX- vs. tumor lysate-loaded DC as vaccines in WEHI3 myeloid leukemia and RENCA bearing mice.	Balb/c mice were challenged intravenously or subcutaneously with WEHI3B and RENCA cells, and then were vaccinated with DC-lysate, DC-TEX and TEX alone. (200 µg/mouse)	WEHI3B-TEX showed the marker profile of WEHI3B tumor cells with higher level of exosome markers, chemokines, and receptors. Delayed tumor growth and prolonged survival rate were observed in all vaccinated mice. The therapeutic efficacy was significantly stronger with more Th and CTL activation in DC-TEX compared to DC-lysate. General applicability of DC-TEX was confirmed by the RENCA tumor-bearing mice.	12
- UNKC6141 pancreatic adenocarcinoma cell line (UNKC)	Ultracentrifugation/sucrose gradient	Strengthen of the therapeutic efficacy of DC-TEX vaccination by blocking of MDSC maturation and activation in pancreatic cancer (PaCa).	UNKC-bearing mice (subcutaneously or orthotopic) after AGS (ATRA, Gemcitabine and Sunitinib) application were immunized intravenously with TEX-loaded DC (DC-TEX).	UNKC-derived TEXs expressed PaCa markers and several common tumor markers. DC-TEX vaccination prolonged the survival rate and delayed the onset of tumor growth as well as reduced UNKC dissemination. Drug combination strengthened DC-TEX efficacy through reduction of MDSCs in vaccinated mice.	152
-Murine breast cancer 4T1 cell line	Gradient ultracentrifugation	Modification of miRNA content of TEXs for enhancement of DC maturation and effector cells activation.	TEX loaded with mimics of miRNAs (miR-155, miR-142, and let-7i) to induce potent dendritic cells (DC) and improve immune stimulation.	Manipulated TEXs enhanced expression of associated markers to DC maturation (MHCII, CD80, and CD40) in both protein and mRNA level and induced CTL activity.	153
-Murine melanoma B16BL6 cell line	Ultracentrifugation	Co-delivery of tumor antigens and adjuvant by genetic modification of exosomes.	B16BL6 cells were transfected with immunostimulatory CpG DNA and then their derived TEXs were utilized as a vaccine in melanoma bearing mice.	CpG DNA-modified TEX enhanced tumor antigen presentation capacity and cytokine release of DCs. More potent cellular and humoral immune response was observed in vaccinated mice model with modified TEXs as compared to unmanipulated TEXs.	154
-HEK293 cell line	Ultracentrifugation	Direct vaccination of murine models with antitumor let7a microRNA and GE11 binding peptide-modified TEX.	Let-7a – GE11 containing TEX were administrated in human tumor xenograft models.	Genetically engineered TEXs conferred greater immunogenicity and inhibition of tumor development in vivo.	155
-AB1 mouse mesothelioma cell line	Ultracentrifugation	Direct comparison of TEX-DC and lysate-DC vaccination in mesothelioma cancer model.	Mesothelioma bearing mice were treated with TEX-loaded DC and lysate-loaded DC vaccines.	Significant reduction in tumor growth and increased survival rate was observed in vaccinated mice with TEX-loaded DC when compared to mice treated with tumor lysate-loaded DC vaccine.	156
-Autologous glioma tumor cells (from patients with glioblastoma multiform)	Ultrafiltration/centrifugation/sucrose Gradient/ultracentrifugation	Assessment of the capacity of autologous glioma tumor cells-derived exosomes in CTLs activity induction compare to tumor lysate.	DCs were pulsed with glioma cells and their derived TEXs, and in vitro CTL activity was then evaluated.	TEXs expressed high level of MHC-I, HSP70, ICAM-1, and MAGE-1 molecules compared with tumor lysate. TEX-loaded DC was more efficient than tumor lysate-loaded DC in induction of glioma specific CTLs.	157
-Myeloma J558 cell line expressing P1A tumor antigen	Ultracentrifugation/sucrose gradient	The comparison of immune responses induced by EXOHSP expressing membrane-bound HSP70 (TEXHSP) or EXO expressing cytoplasmic HSP70 (TEXHS).	Myeloma J558 cell line were transfected by membrane-bound inducible HSP70 expressing vectors and then myeloma bearing mice were treated with TEXHSP or TEXHS pulsed DCs.	TEXHSP-loaded DC efficiently stimulated DC maturation and tumor-specific CD4+Th1, CD8+ CTL, and NK cells responses in tumor-bearing mice. TEXHSP-loaded DC more strongly inhibited tumor growth and increased survival in vaccinated mice compared with TEXHS-loaded DC.	158
-MUC1-expressing CT26 cell line	Ultracentrifugation/sucrose gradient	Enhancement of the immune-stimulating activity of TEXs by heat treatment of tumor cells.	MUC1-CT26 cells were exposed to heat shock, then tumor bearing mice were vaccinated by HS-TEX or untreated TEX.	HS-TEX elicited Th1 cells activation through increased IgG2a and IFN-γ production. HS-TEX strongly suppressed tumor growth in autologous and allogeneic mouse models compared with untreated TEX.	159
-CT26 murine colon carcinoma cell line -TA3HA murine mammary carcinoma cell line	Ultracentrifugation/sucrose gradient	Genetic manipulation of TEXs for tumor antigens delivery.	Immunogenic potential of MUC-containing exosomes was tested in autogenic and allogeneic tumors.	MUC-containing TEXs were capable to induce DC maturation and IFN-γ secretion as well as greater inhibition of tumor growth in both allogeneic and autologous tumor-bearing mice compared to non-MUC1-containing TEXs.	24
-EG7 expressing ovalbumin cell line (OVA- transfected EL4 mouse thymoma cell line)	Ultracentrifugation/sucrose gradient	Protein transfer of SEA on TEXs in order to increase their immunogenicity.	C57BL/6 mice were immunized subcutaneously with TEX, SEA-TEX or mixture of TEX and SEA, and were challenged with EG7-OVA cells.	SEA-TEX more efficiently induced tumor-specific CD 8+T, NK, and CD4+ T cells, and increased IL-2 and IFN-γ production compared to TEX or mixture of TEX and SEA. SEA-TEX elicited greater immune response in tumor-bearing mice.	160
-L1210 murine B lymphocytic leukemia cell line	Ultrafiltration Centrifugation/sucrose gradient/ultracentrifugation	Assessment of L1210-derived TEXs impact as a prophylactic vaccine in syngeneic mice models.	DBA/2 mice were subcutaneously immunized with L1210-TEXs (2.5 or 5 µg) and then challenged with L1210 leukemia cells.	Mice treated with L1210-TEXs showed tumor growth suppression. L1210-TEXs stimulated in vitro tumor-specific CTL responses.	149
-Fon (HLA-A2+) and Mel-888 (HLA-A2−) human melanoma tumor cell lines	Ultracentrifugation/sucrose gradient	The comparison of immune responses induced by TEXs and tumor cells lysate in prophylactic and therapeutic mice models.	Mice models were immunized by different doses of TEXs and tumor cell-derived lysate as alone or loaded on DC.	More protective antitumor activity was observed significantly in syngeneic and allogeneic mice models which treated with TEXs when compared to mice treated with tumor lysate. Lower doses of TEXs were more potent in suppressing of tumor growth compared to tumor cell lysate.	13
-AB1 mouse mesothelioma cell line	Ultracentrifugation	Study of the impact of antigen source in cancer immunotherapy outcome.	Mesothelioma bearing mice were treated with DCs loaded by different antigen sources (tumor cell line lysate, ex vivo tumor lysate and tumor cell line-derived TEX).	Although DC-EXO was effective in prolonging overall survival, but tumor cell line lysate-pulsed DCs were more effective.	161
- B16-OVA and B16 melanoma cell lines	sucrose gradient/ultracentrifugation	Evaluation of the effect of VSV-G incorporation in TEXs on their immunogenicity.	Melanoma cell lines B16-OVA and B16 were transduced by VSV-G-expressing vectors and then melanoma bearing mice were treated with VSV-G containing TEXs loaded on DC.	VSV-G improved TEXs antigenic transfer to DCs and more potently stimulated phenotypic and functional maturation of DCs. VSV-G bearing TEXs elicited strong in vivo antitumor CTL responses.	162
-E.G7-OVA tumor cells expressing Ovalbumin (OVA)	Serial centrifugation/sucrose gradients/ultracentrifugation	Further improvement of TEX-based vaccination through expression of IL-2 in them.	E.G7-OVA tumor cells were transfected with IL-2 gene and then tumor bearing mice were immunized with TEX/IL-2, TEX+IL-2 and TEX alone.	TEX/IL-2 conferred more potent CTL, NK, and CD4+ T cell activation, also significant suppression of tumor growth compared to TEX+IL-2 or TEX. TEX/IL-2 increased IL-2 and IFN-γ production in vaccinated mice.	163
-Human RC-2 renal cancer cell line	Ultracentrifugation	Improving TEX immunogenicity by IL-12 gene modification in tumor cells.	Human renal tumor cells were genetically modified with IL-12 gene and then in vitro immune responses of TEX/IL-12 derived from these cells were examined.	TEX/IL-12 activated T-cell proliferation and increased release of IFN-γ. TEX/IL-12 induced in vitro antigen-specific cytotoxic effect.	164
-B16F1 melanoma cell line	Ultracentrifugation	In vitro assessment of immunological activity of melanoma cell-derived TEXs (mcd-TEXs).	Immunogenic potential of mcd-TEXs was tested in vitro.	Mcd-TEXs could stimulate in vitro CD4+ T cell proliferation and NF-kB activation in macrophages.	83
- Hepa1-6 murine hepatocellular carcinoma (HCC) cell line -Human HepG2 and Hep3B HCC cell lines	Ultracentrifugation	Human in vitro and murine in vivo study of immune efficacy of HCC cell line-derived TEXs compare to cell lysate.	-For murine in vivo study, C57BL6 mice were immunized subcutaneously with PBS, TEX-pulsed DC (30 µg/mouse), lysate-pulsed DC in therapeutic and preventive as well as ectopic and orthotopic models. -For human in vitro study, DCs were pulsed with TEXs and then in vitro CTL activity was evaluated.	Immunization with TEX-pulsed DC exhibited stronger in vivo antitumor effects and decreased TNF-β and IL-10 levels in tumor sites compared to lysate-pulsed DC. TEX-induced efficient in vitro human dendritic cells maturation and showed HCC-specific cytolysis as well as cross-protective effects against pancreatic cancer cells.	165
-B16BL6 melanoma cell line -Murine melanoma tumor	Homogenization/sonication/ultracentrifugation	Assessment of the therapeutic effect of autologous TEXs in melanoma tumor and metastatic mice models.	Autologous murine melanoma tumors were dissected and homogenized, and then immunogenicity of their isolated TEXs was evaluated in vitro and in vivo.	TEXs plus poly I:C (as adjuvant) increased secretion of IL-12p70 and Th1-related antibody, IgG2a. TEXs vaccination exhibited strong antitumor effects in primary and metastatic in vivo cancer models.	139
- Hepa1-6 cell line	Ultracentrifugation	In vivo evaluation of the effect of DC-TEX and sorafenib combination on treatment outcome.	Orthotopic HCC mice were vaccinated with different combinations of DC-TEX, sorafenib and PD-1 Ab, and then in vivo immune responses were evaluated.	DC-TEX activated CD8+T cells and decreased Treg cells. DC-TEX conferred more PD-1+CD8+T cells in orthotopic HCC mice. Combination of sorafenib, PD-1 Ab and DC-TEX promoted the treatment outcomes in vaccinated mice.	166