Additional file 1 of Engineered exosomes as an in situ DC-primed vaccine to boost antitumor immunity in breast cancer

Additional file 1: Figure S1. Electroporation protocol optimization and targeting of HELA-Exos. (A and B) FITC-labeled ELANE or CX-rhodamine-labeled Hiltonol dispersed in pure water at various concentrations was applied for calibration. The fluorescence standard curve of ELANE or Hiltonol was generated by measuring the fluorescence intensity at 519 nm and 597 nm. (C and D) FITC-labeled ELANE and CX-Rhodamine-labeled Hiltonol were electroporated with exosomes in 200 µl of buffer at the settings shown on the x-axis, and ELANE and Hiltonol loading was determined based on the fluorescence standard curve. The data are presented as the mean ± SD; n = 3. (E) The cellular uptake of MDA-MB-231 cells or A549 cells after incubation with Texs-DiI or HELA-Exos-DiI for 2 h was evaluated by CLSM. Blue: DAPI. Red: DiI. Scale bar, 25 μm. CLSM: confocal laser scanning microscopy. Figure S2. Apoptosis analysis and specific cytotoxic activity of CD8+ T cells. (A) Gating strategy for analysis of MDA-MB-231 cells; the percentages of PI-positive cells were measured. (B) Apoptosis analysis. Images of western blots for cleaved CASP3/CASP3 and cleaved PARP1/PARP1 in MDA-MB-231 cells. (C) CD8+ T cells were harvested from various treatment groups and incubated with target cells (MDA-MB-231, MCF7, A549, and MCF10A cells), and cytotoxicity was detected with a CCK-8 kit. The data are presented as the mean ± SD; n = 6. t test and one-way ANOVA were performed for statistical analysis (****: P < 0.0001; *: P < 0.05; ns: P > 0.05). Figure S3. Fluorescence imaging, pharmacokinetic curves, and safety of HELA-Exos in vivo. (A) In vivo fluorescence imaging in orthotopic MDA-MB-231 tumor-bearing mice at 2, 12, and 24 h following injection with CX-Rhodamine-labeled Hiltonol and HELA-Exos-DiI. (B) In vivo pharmacokinetic curves of Hiltonol and HELA-Exos. (C) Pathological examination of the major organs (the lung, heart, liver, spleen, and kidney). The tissue sections were stained with H&E. Scale bar, 100 μm. (D) The levels of the serum biochemical parameters ALT, AST, BUN, and CREA. The data are presented as the mean ± SD; n = 6. One-way ANOVA was performed for statistical analysis (ns: P > 0.05). Figure S4. APCs were recruited to the tumor microenvironment after HELA-Exo treatment. Balb/c mice with orthotopic breast cancer were sacrificed at day 30 after treatment initiation. Tumor-infiltrating DCs (CD11c+) and macrophages (F4/80+) were detected by IF. Blue: DAPI; Green: F4/80; Red: CD11c. Scale bar, 25 μm. IF: immunofluorescence. Figure S5. Specific cytotoxic activity of CD8+ T cells from the tumor microenvironment of Balb/c mice with orthotopic breast cancer. Balb/c mice with orthotopic breast cancer were sacrificed at day 30 after treatment initiation. The tumor tissue was cut into small pieces to generate a single-cell suspension. CD8+ T cells were sorted and incubated with target cells (MDA-MB-231, MCF7, A549, and MCF10A cells), and cytotoxicity was detected with a CCK-8 kit. The data are presented as the mean ± SD; n = 6. A t test and one-way ANOVA were performed for statistical analysis (****: P < 0.0001; **: P < 0.01; ns: P > 0.05). Figure S6. Reduced systemic inflammatory toxicity of HELA-Exos. Balb/c mice with orthotopic breast cancer were sacrificed at day 30 after treatment initiation. The serum levels of the cytokines IL-6, IL-12, and TNF-α. The data are presented as the mean ± SD; n = 6. A t test was performed for statistical analysis (****: P < 0.0001; ns: P > 0.05). Figure S7. DC depletion efficiency in Balb/c mice and specific cytotoxic activity of CD8+ T cells. (A) Gating strategy for the analysis of DCs; the percentages of CD11c+ DCs in the lymph nodes and spleen were measured. (B) DC-depleted or nondepleted Balb/c mice with orthotopic breast cancer were sacrificed at Day 30 after treatment initiation. The tumor tissue was cut into small pieces to generate a single-cell suspension. CD8+ T cells were sorted and incubated with target cells (MDA-MB-231, MCF7, A549, and MCF10A cells), and cytotoxicity was detected with a CCK-8 kit. The data are presented as the mean ± SD; n = 6. A t test and one-way ANOVA were performed for statistical analysis (****: P < 0.0001; *: P < 0.05; ns: P > 0.05). Figure S8. Tumor organoids were established from tumor tissue, and PBMCs were isolated from peripheral blood before the start of coculture. On the day of coculture, organoids were isolated from Geltrex, dissociated into single cells and plated together with PBMCs in the presence of vaccines. After 1 week of coculture, PBMCs were re-stimulated with tumor cells from organoids. After two weeks of coculture, immune infiltration in organoids was evaluated by flow cytometry and IF, and the growth-inhibitory effects in organoids of HELA-Exos were evaluated by a viability assay and live/dead cell staining.

附加文件1：
图S1 电穿孔方案优化与HELA-Exos靶向修饰。(A和B) 将不同浓度的异硫氰酸荧光素（FITC）标记的ELANE或四甲基异硫氰酸罗丹明（CX-rhodamine）标记的Hiltonol分散于纯水中进行校准，通过在519 nm和597 nm处测定荧光强度，绘制ELANE或Hiltonol的荧光标准曲线。(C和D) 将FITC标记的ELANE与CX-罗丹明（CX-Rhodamine）标记的Hiltonol与外泌体在200 μL缓冲液中按照横轴所示参数进行电穿孔，基于荧光标准曲线计算ELANE和Hiltonol的载药量。数据以平均值±标准差（SD）表示，n=3。(E) 将MDA-MB-231细胞或A549细胞与Texs-DiI或HELA-Exos-DiI共孵育2 h后，通过共聚焦激光扫描显微镜（confocal laser scanning microscopy, CLSM）评估细胞摄取情况。蓝色：4',6-二脒基-2-苯基吲哚（DAPI）；红色：DiI。标尺：25 μm。
图S2 CD8+ T细胞凋亡分析与特异性细胞毒活性。(A) MDA-MB-231细胞的设门分析策略，检测碘化丙啶（PI）阳性细胞百分比。(B) 凋亡分析：MDA-MB-231细胞中剪切型CASP3/CASP3及剪切型PARP1/PARP1的蛋白质免疫印迹图像。(C) 从各处理组中收获CD8+ T细胞，与靶细胞（MDA-MB-231、MCF7、A549及MCF10A细胞）共孵育，采用CCK-8试剂盒检测细胞毒性。数据以平均值±标准差表示，n=6。采用t检验及单因素方差分析（one-way ANOVA）进行统计学分析（****：P < 0.0001；*：P < 0.05；ns：P > 0.05）。
图S3 HELA-Exos的体内荧光成像、药代动力学曲线与安全性。(A) 向原位MDA-MB-231荷瘤小鼠注射CX-罗丹明标记的Hiltonol与HELA-Exos-DiI后，分别于2、12、24 h进行体内荧光成像。(B) Hiltonol与HELA-Exos的体内药代动力学曲线。(C) 主要脏器（肺、心、肝、脾、肾）的病理学检查，组织切片经苏木精-伊红（H&E）染色。标尺：100 μm。(D) 血清生化指标丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、尿素氮（BUN）及肌酐（CREA）水平。数据以平均值±标准差表示，n=6。采用单因素方差分析进行统计学分析（ns：P > 0.05）。
图S4 HELA-Exos治疗后抗原呈递细胞（APCs）招募至肿瘤微环境。于治疗开始后第30天处死原位乳腺癌Balb/c小鼠，采用免疫荧光（IF, immunofluorescence）检测肿瘤浸润性树突状细胞（CD11c+）与巨噬细胞（F4/80+）。蓝色：DAPI；绿色：F4/80；红色：CD11c。标尺：25 μm。
图S5 原位乳腺癌Balb/c小鼠肿瘤微环境中CD8+ T细胞的特异性细胞毒活性。于治疗开始后第30天处死原位乳腺癌Balb/c小鼠，将肿瘤组织剪碎制备单细胞悬液，分选CD8+ T细胞并与靶细胞（MDA-MB-231、MCF7、A549及MCF10A细胞）共孵育，采用CCK-8试剂盒检测细胞毒性。数据以平均值±标准差表示，n=6。采用t检验及单因素方差分析进行统计学分析（****：P < 0.0001；**：P < 0.01；ns：P > 0.05）。
图S6 HELA-Exos降低全身性炎症毒性。于治疗开始后第30天处死原位乳腺癌Balb/c小鼠，检测血清中细胞因子白细胞介素-6（IL-6）、白细胞介素-12（IL-12）及肿瘤坏死因子-α（TNF-α）水平。数据以平均值±标准差表示，n=6。采用t检验进行统计学分析（****：P < 0.0001；ns：P > 0.05）。
图S7 Balb/c小鼠树突状细胞清除效率及CD8+ T细胞特异性细胞毒活性。(A) 树突状细胞分析设门策略，检测淋巴结及脾脏中CD11c+树突状细胞的百分比。(B) 于治疗开始后第30天处死经树突状细胞清除或未清除的原位乳腺癌Balb/c小鼠，将肿瘤组织剪碎制备单细胞悬液，分选CD8+ T细胞并与靶细胞（MDA-MB-231、MCF7、A549及MCF10A细胞）共孵育，采用CCK-8试剂盒检测细胞毒性。数据以平均值±标准差表示，n=6。采用t检验及单因素方差分析进行统计学分析（****：P < 0.0001；*：P < 0.05；ns：P > 0.05）。
图S8 从肿瘤组织中构建肿瘤类器官，并于共培养开始前从外周血中分离外周血单个核细胞（PBMCs）。共培养当日，从Geltrex基质胶中分离类器官，消化为单细胞后与外周血单个核细胞及疫苗共同铺板。共培养1周后，用类器官来源的肿瘤细胞重新刺激外周血单个核细胞。共培养2周后，采用流式细胞术及免疫荧光检测类器官中的免疫浸润情况，并通过活性检测试剂盒与活死细胞染色评估HELA-Exos对类器官的生长抑制效应。