Additional file 2 of A new histone deacetylase inhibitor remodels the tumor microenvironment by deletion of polymorphonuclear myeloid-derived suppressor cells and sensitizes prostate cancer to immunotherapy

Additional file 2: Fig. S1. CN133 did not change the infiltration of CD4 and CD8 T cells in RM1 subcutaneous tumors. A. Flow cytometric data showing resulting of CD4+ and CD8+ T cells in spleens of CN133 and placebo groups at day 25. B. Fluorescent images of CD3+CD8+ T cells in placebo and CN133 treatment of RM1 subcutaneous tumors from C56BL/6J mice. Scale bar, 50 μm. Fig. S2. Combination of CN133 with anti-PD-1 inhibit PCa growth and improve the survival percentage in FVB mice bearing MyC-CaP subcutaneous tumors. Synergistic effect of CN133 with anti-PD-1 enhanced CD8 T+ cell population and function, reduced the secretion of Arg-1 and iNOS in C57BL/J6 models. A. Xenografts tumor volume of FVB mice with MyC-CaP prostate cancer cell line treated with placebo, CN133, anti-PD-1 (1mg/kg) or CN133 (1mg/kg) combined with anti-PD-1. B. Survival percentage of FVB subcutaneous PCa mice models were assessed after treatment of placebo, CN133, anti-PD-1 or CN133 plus anti-PD-1. C. BLI intensity of subcutaneous tumors of FVB mice after treatments of Placebo, CN133, anti-PD-1 or CN133 plus anti-PD-1. D and E. Flow cytometric analyses examined positive percentages of Arg-1 and iNOS in the PMN-MDSCs (D), and granzyme B and perforin in the CD8+ T cells (E) in spleens of placebo, CN133, anti-PD-1 or CN133 plus anti-PD-1, treatment of subcutaneous C57BL/J6 PCa mice. Fig. S3. CN133 combination of PD-1 did not improve CD8 T cell population and function on the FVB mice bearing murine RM1 bone metastatic PCa tumors. A. MRI images of tumor-bearing bones in mice treated with PD-1 or PD-1 plus CN133 at day 25. B and C. CD4 and CD8 T cells (B), and granzyme B and perforin effector cytokine (C) were quantified by flow cytometric analyses in the bone marrow of this PCa mice models at day 25.

附加文件2：图S1。CN133不改变RM1皮下瘤中CD4和CD8 T细胞的浸润情况。A. 流式细胞术数据展示了第25天时CN133组与安慰剂组小鼠脾脏中CD4+和CD8+ T细胞的占比情况。B. C56BL/6J小鼠RM1皮下瘤经安慰剂与CN133处理后，其CD3+CD8+ T细胞的荧光成像图。比例尺：50 μm。
图S2。CN133联合抗PD-1可抑制前列腺癌（prostate cancer, PCa）生长，并提高携带MyC-CaP皮下瘤的FVB小鼠的存活率。CN133与抗PD-1的协同作用可增加CD8+ T细胞的数量与功能，并降低C57BL/J6模型中精氨酸酶1（Arg-1）与诱导型一氧化氮合酶（iNOS）的分泌水平。A. 接种MyC-CaP前列腺癌细胞系的FVB小鼠分别经安慰剂、CN133、抗PD-1（1mg/kg）或CN133（1mg/kg）联合抗PD-1处理后的异种移植瘤体积。B. 评估经安慰剂、CN133、抗PD-1或CN133联合抗PD-1处理后的FVB皮下前列腺癌小鼠模型的存活率。C. FVB小鼠经上述四种处理后皮下瘤的生物发光成像（BLI）强度。D与E：流式细胞术分析检测了经安慰剂、CN133、抗PD-1或CN133联合抗PD-1处理的皮下C57BL/J6前列腺癌小鼠脾脏中，多形核髓系来源抑制细胞（PMN-MDSCs）内Arg-1与iNOS的阳性百分比（D），以及CD8+ T细胞内颗粒酶B与穿孔素的阳性百分比（E）。
图S3。CN133联合PD-1治疗无法提升携带RM1小鼠骨转移性前列腺癌的FVB小鼠体内CD8+ T细胞的数量与功能。A. 第25天时，经PD-1或PD-1联合CN133处理的小鼠荷瘤骨的磁共振成像（MRI）图。B与C：第25天时，通过流式细胞术定量分析该前列腺癌小鼠模型骨髓中的CD4与CD8 T细胞（B），以及颗粒酶B与穿孔素这类效应细胞因子（C）的水平。