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Radiation-induced small extracellular vesicles as “carriages” promote tumor antigen release and trigger antitumor immunity

Lin, Wanzun, Xu, Yanyan, Chen, Xiaochuan, Liu, Jun, Weng, Youliang, Zhuang, Qingyang, Lin, Feifei, Huang, Zongwei, Wu, Shihong, Ding, Jianming, Chen, Long, Qiu, Xianxin, Zhang, Lurong, Wu, Junxin, Lin, Duo, Qiu, Sufang·2020·Theranostics
DOI 10.7150/thno.43539PMC PMC7163438
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Figure 1 Characterization of small extracellular vesicles (sEVs). (A) Particle size distribution measured via nanoparticle tracking analysis (NTA). (B) sEV morphology revealed via transmission electron microscopy (TEM). (C) Western blot analysis of specific sEV-specific surface markers.
Figure 1 Characterization of small extracellular vesicles (sEVs). (A) Particle size distribution measured via nanoparticle tracking analysis (NTA). (B) sEV morphology revealed via transmission electron microscopy (TEM). (C) Western blot analysis of specific sEV-specific surface markers.CC BY · PubMed Central
Figure 2 Antitumor immunity triggered by radiation-induced sEVs against primary tumor and experimental lung metastasis. (A) Experimental schema depicting sEV purification and immunization. H22 cells and 4T1 cells were subjected to 8 Gy irradiation, and sEVs derived from 8 Gy-irradiated cells (8 Gy-sEVs) or non-irradiated cells (0 Gy-sEVs) were purified via ultracentrifugation. ICR and BALB/c mice were immunized with 200 μg of each type of sEV four times at 5-day intervals and challenged by injection of tumor cells. (B) Cell-cell contact between green fluorescent protein (GFP)-positive 4T1 cells and lymphocytes was imaged under fluorescent reverse-microscopy. In a co-culture of 4T1 tumor cells and lymphocytes, 4T1 tumor cells were closely surrounded by lymphocytes from mice immunized with 8 Gy-sEVs, although this was not observed in lymphocytes from mice immunized with 0 Gy-sEVs or the control group. The killing effect was measured as the amount of lactate dehydrogenase (LDH) released (n = 4/group). (C) In vivo bioluminescence imaging. GFP-luciferase-4T1 cells were intravenously injected into the tail vein of BALB/c mice after immunization. Two weeks later, the growth of 4T1 experimental lung metastases was measured using an IVIS Lumina III in vivo imaging system. Luciferase signals from mice immunized with 8 Gy-sEVs were lower than those obtained from the control or 0 Gy group (n = 6/group). (D) BALB/c mice were intravenously injected with 4T1 cells via the tail vein. Differences in survival were observed between BALB/c mice immunized with 0 Gy-sEVs, 8 Gy-sEVs, and the control group (n = 10/group). (E) Images and hematoxylin & eosin staining of lung metastases and statistical analysis of lung metastatic nodules (n = 6/group). (F-G) Primary tumor growth and growth curve of 4T1 breast cancer cells (n = 6/group). (H) Tumor weights of 4T1 breast cancer cells at the end of the experiment (n = 6/group). (I) Primary tumor growth and growth curve of H22 hepatoma cells (n = 6/group). (J) Tumor weights of H22 hepatoma cells at the end of the experiment (n = 6/group). P-values were determined using a two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 2 Antitumor immunity triggered by radiation-induced sEVs against primary tumor and experimental lung metastasis. (A) Experimental schema depicting sEV purification and immunization. H22 cells and 4T1 cells were subjected to 8 Gy irradiation, and sEVs derived from 8 Gy-irradiated cells (8 Gy-sEVs) or non-irradiated cells (0 Gy-sEVs) were purified via ultracentrifugation. ICR and BALB/c mice were immunized with 200 μg of each type of sEV four times at 5-day intervals and challenged by injection of tumor cells. (B) Cell-cell contact between green fluorescent protein (GFP)-positive 4T1 cells and lymphocytes was imaged under fluorescent reverse-microscopy. In a co-culture of 4T1 tumor cells and lymphocytes, 4T1 tumor cells were closely surrounded by lymphocytes from mice immunized with 8 Gy-sEVs, although this was not observed in lymphocytes from mice immunized with 0 Gy-sEVs or the control group. The killing effect was measured as the amount of lactate dehydrogenase (LDH) released (n = 4/group). (C) In vivo bioluminescence imaging. GFP-luciferase-4T1 cells were intravenously injected into the tail vein of BALB/c mice after immunization. Two weeks later, the growth of 4T1 experimental lung metastases was measured using an IVIS Lumina III in vivo imaging system. Luciferase signals from mice immunized with 8 Gy-sEVs were lower than those obtained from the control or 0 Gy group (n = 6/group). (D) BALB/c mice were intravenously injected with 4T1 cells via the tail vein. Differences in survival were observed between BALB/c mice immunized with 0 Gy-sEVs, 8 Gy-sEVs, and the control group (n = 10/group). (E) Images and hematoxylin & eosin staining of lung metastases and statistical analysis of lung metastatic nodules (n = 6/group). (F-G) Primary tumor growth and growth curve of 4T1 breast cancer cells (n = 6/group). (H) Tumor weights of 4T1 breast cancer cells at the end of the experiment (n = 6/group). (I) Primary tumor growth and growth curve of H22 hepatoma cells (n = 6/group). (J) Tumor weights of H22 hepatoma cells at the end of the experiment (n = 6/group). P-values were determined using a two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).CC BY · PubMed Central
Figure 3 CD8 and CD4 cell infiltration in 8 Gy-sEV-immunized mice. (A-C) The lymph nodes, spleen, and blood of BALB/c mice were double-stained using anti-mouse fluorescein isothiocyanate (FITC)-anti-mouse CD3, peridinin chlorophyll protein complex (PerCP)-anti-mouse CD8, or allophycocyanin (APC)-anti-mouse CD4, and analyzed using flow cytometry to detect CD4 and CD8. (D-F) 4T1 lung metastases, 4T1 primary tumors, and H22 primary tumors were harvested at the end of the experiment and stained with anti-mouse CD8 (imaged at 200× magnification). P-values were determined using a two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 3 CD8 and CD4 cell infiltration in 8 Gy-sEV-immunized mice. (A-C) The lymph nodes, spleen, and blood of BALB/c mice were double-stained using anti-mouse fluorescein isothiocyanate (FITC)-anti-mouse CD3, peridinin chlorophyll protein complex (PerCP)-anti-mouse CD8, or allophycocyanin (APC)-anti-mouse CD4, and analyzed using flow cytometry to detect CD4 and CD8. (D-F) 4T1 lung metastases, 4T1 primary tumors, and H22 primary tumors were harvested at the end of the experiment and stained with anti-mouse CD8 (imaged at 200× magnification). P-values were determined using a two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).CC BY · PubMed Central
Figure 4 Proteomic and bioinformatic analysis of altered cargos in radiation-induced sEVs. (A) Scatter plot showing the distribution of 146 differentially expressed proteins quantified in 8 Gy-sEVs and 0 Gy-sEVs (fold-change > 1.5 and P < 0.05). Red dots indicate up-regulated proteins and green dots indicate down-regulated proteins. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed proteins. (C) Expression of up-regulated proteins in various cancers in the Cancer Genome Atlas (TCGA). The intensity of the color represents log2 fold-change (tumor vs normal). Y-axis shows protein name and X-axis shows tumor type, including lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), kidney renal clear cell carcinoma (KIRC), colon adenocarcinoma (COAD), breast invasive carcinoma (BRCA), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA), bladder urothelial carcinoma (BLCA), kidney renal papillary cell carcinoma (KIRP), prostate adenocarcinoma (PRAD), liver hepatocellular carcinoma (LIHC), and esophageal carcinoma (ESCA).
Figure 4 Proteomic and bioinformatic analysis of altered cargos in radiation-induced sEVs. (A) Scatter plot showing the distribution of 146 differentially expressed proteins quantified in 8 Gy-sEVs and 0 Gy-sEVs (fold-change > 1.5 and P < 0.05). Red dots indicate up-regulated proteins and green dots indicate down-regulated proteins. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed proteins. (C) Expression of up-regulated proteins in various cancers in the Cancer Genome Atlas (TCGA). The intensity of the color represents log2 fold-change (tumor vs normal). Y-axis shows protein name and X-axis shows tumor type, including lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), kidney renal clear cell carcinoma (KIRC), colon adenocarcinoma (COAD), breast invasive carcinoma (BRCA), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA), bladder urothelial carcinoma (BLCA), kidney renal papillary cell carcinoma (KIRP), prostate adenocarcinoma (PRAD), liver hepatocellular carcinoma (LIHC), and esophageal carcinoma (ESCA).CC BY · PubMed Central
Figure 5 Peptides derived from up-regulated proteins in sEVs triggered antitumor immunity. (A) Two peptides derived from CUB domain-containing protein 1 (CDCP1) were synthesized. (B) Experiment design. (C) Flow cytometry (FCM) analysis of differences of CD8+-interferon (IFN)γ+ T lymphocytes in the control group (phosphate-buffered saline (PBS) with Freund's adjuvant) and peptide group (CDCP1 peptides with Freund's adjuvant; n = 3/group). (D) Primary 4T1 tumor sizes in the control group (immunization with PBS four times, at 5-day intervals) and peptide group (immunization with 200 µg CDCP1 peptides four times, at 5-day intervals; n = 6/group). (E) Primary 4T1 tumor growth curve in the control (immunization with PBS four times, at 5-day intervals) and peptide-treated group (immunization with 200 µg CDCP1 peptides four times, at 5-day intervals; n = 6/group). (F) Primary 4T1 tumor weights (n = 6/group). (G-H) Experimental metastasis model and in vivo imaging of the lungs in the control group (immunization with PBS four times, at 5-day intervals) and peptide group (immunization with 200 µg CDCP1 peptides four times, at 5-day intervals; n = 6/group). P-values were determined using a two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 5 Peptides derived from up-regulated proteins in sEVs triggered antitumor immunity. (A) Two peptides derived from CUB domain-containing protein 1 (CDCP1) were synthesized. (B) Experiment design. (C) Flow cytometry (FCM) analysis of differences of CD8+-interferon (IFN)γ+ T lymphocytes in the control group (phosphate-buffered saline (PBS) with Freund's adjuvant) and peptide group (CDCP1 peptides with Freund's adjuvant; n = 3/group). (D) Primary 4T1 tumor sizes in the control group (immunization with PBS four times, at 5-day intervals) and peptide group (immunization with 200 µg CDCP1 peptides four times, at 5-day intervals; n = 6/group). (E) Primary 4T1 tumor growth curve in the control (immunization with PBS four times, at 5-day intervals) and peptide-treated group (immunization with 200 µg CDCP1 peptides four times, at 5-day intervals; n = 6/group). (F) Primary 4T1 tumor weights (n = 6/group). (G-H) Experimental metastasis model and in vivo imaging of the lungs in the control group (immunization with PBS four times, at 5-day intervals) and peptide group (immunization with 200 µg CDCP1 peptides four times, at 5-day intervals; n = 6/group). P-values were determined using a two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).CC BY · PubMed Central
Figure 6 Heat shock protein (HSP)70 and HSP90 expression in radiation-enriched sEVs. (A) Proteomic analysis showing up-regulation of the HSP family in sEVs from irradiated 4T1 cells. (B) Western blot confirming that Hsp70 and Hsp90 were up-regulated in sEVs from irradiated 4T1 cells.
Figure 6 Heat shock protein (HSP)70 and HSP90 expression in radiation-enriched sEVs. (A) Proteomic analysis showing up-regulation of the HSP family in sEVs from irradiated 4T1 cells. (B) Western blot confirming that Hsp70 and Hsp90 were up-regulated in sEVs from irradiated 4T1 cells.CC BY · PubMed Central
Figure 7 the underlying mechanism on how sEVs achieve antitumor immunity after radiation. (A) Experimental design. Bone marrow cells were isolated from the femurs of mice and stimulated with GM-CSF and IL-4 for DCs differentiation. Then, DCs were pulsed with sEVs and co-culture with CD8+ T cell. Western bolt was used to analyze activated signaling in CD8+ T cell. (B) Detection of CD11c+ dendritic cells by FCM after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. (C) Uptake of sEVs by DCs. sEVs were first stained with PKH67 dye before 24-h culture with DCs and analysis via confocal microscopy. (D) Panels illustrate the gating strategy used for isolation of CD8+ T cell subsets via fluorescence-activated cell sorting. (E) Western blot revealed the activated signaling in CD8+ T cell.
Figure 7 the underlying mechanism on how sEVs achieve antitumor immunity after radiation. (A) Experimental design. Bone marrow cells were isolated from the femurs of mice and stimulated with GM-CSF and IL-4 for DCs differentiation. Then, DCs were pulsed with sEVs and co-culture with CD8+ T cell. Western bolt was used to analyze activated signaling in CD8+ T cell. (B) Detection of CD11c+ dendritic cells by FCM after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. (C) Uptake of sEVs by DCs. sEVs were first stained with PKH67 dye before 24-h culture with DCs and analysis via confocal microscopy. (D) Panels illustrate the gating strategy used for isolation of CD8+ T cell subsets via fluorescence-activated cell sorting. (E) Western blot revealed the activated signaling in CD8+ T cell.CC BY · PubMed Central
Figure 8 Mechanism by which radiation-induced sEVs trigger antitumor immunity.
Figure 8 Mechanism by which radiation-induced sEVs trigger antitumor immunity.CC BY · PubMed Central

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Authors
Lin, W., Xu, Y., Chen, X., Liu, J., Weng, Y., Zhuang, Q., Lin, F., Huang, Z., Wu, S., Ding, J., Chen, L., Qiu, X., Zhang, L., Wu, J., Lin, D., & Qiu, S.
Venue
Theranostics
Publisher
Ivyspring International Publisher
Published
2020
Volume
10
Issue
11
Pages
4871-4884
ISSN
1838-7640
DOI
10.7150/thno.43539
PMC
PMC7163438
Cited by
97
References
0
Language
en

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