Human pancreatic ductal adenocarcinoma (PDAC) responds poorly to immune checkpoint inhibitor (ICPi). While the mechanism is not completely clear, it has been recognized that tumor microenvironment (TME) plays key roles. We investigated if targeting CD47 with a monoclonal antibody could enhance the response of PDAC to ICPi by altering the TME. Using immunohistochemistry, we examined tumor-infiltrating CD68+ pan-macrophages (CD68+ M) and CD163+ M2 macrophages (CD163+ M2) and tumor expression of CD47 and PD-L1 proteins in 106 cases of PDAC. The efficacy of CD47 blockade was examined in xenograft models. CD45+ immune cells from syngeneic tumor models were subjected to single-cell RNA-sequencing (scRNA-seq) by using the 10x Genomics pipeline. We found that CD47 expression correlated with the level of CD68+ M but not CD163+ M2. High levels of tumor-infiltrating CD68+ M, CD163+ M2, and CD47 expression were significantly associated with worse survival. CD47high/CD68+ Mhigh and CD47high/CD163+ M2high correlated significantly with shorter survival, whereas CD47low/CD68+ Mlow and CD47low/CD163+ M2low correlated with longer survival. Intriguingly, CD47 blockade decreased the tumor burden in the Panc02 but not in the MPC-83 syngeneic mouse model. Using scRNA-seq, we showed that anti-CD47 treatment significantly remodeled the intratumoral lymphocyte and macrophage compartments in Panc02 tumor-bearing mice by increasing the pro-inflammatory macrophages that exhibit anti-tumor function, while reducing the anti-inflammatory macrophages. Moreover, CD47 blockade not only increased the number of intratumoral CD8+ T cells, but also remodeled the T cell cluster toward a more activated one. Further, combination therapy targeting both CD47 and PD-L1 resulted in synergistic inhibition of PDAC growth in the MPC-83 but not in Panc02 model. MPC-83 but not Panc02 mice treated with both anti-CD47 and anti-PD-L1 showed increased number of PD-1+CD8+ T cells and enhanced expression of key immune activating genes. Our data indicate that CD47 targeting induces compartmental remodeling of tumor-infiltrating immune cells of the TME in PDAC. Different PDAC mouse models exhibited differential response to the anti-CD47 and anti-PD-L1 blockade due to the differential effect of this combination treatment on the infiltrating immune cells and key immune activating genes in the TME established by the different PDAC cell lines.

中文翻译：

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单细胞RNA测序揭示胰腺癌中由抗CD47靶向诱导的肿瘤浸润免疫细胞的区室重塑。

人胰导管腺癌（PDAC）对免疫检查点抑制剂（ICPi）的反应较差。虽然机理尚不完全清楚，但已经认识到肿瘤微环境（TME）发挥着关键作用。我们研究了用单克隆抗体靶向CD47是否可以通过改变TME增强PDAC对ICPi的反应。使用免疫组织化学，我们检查了106例PDAC中浸润肿瘤的CD68 +巨噬细胞（CD68 + M）和CD163 + M2巨噬细胞（CD163 + M2）以及CD47和PD-L1蛋白的肿瘤表达。在异种移植模型中检查了CD47阻断的功效。使用10x Genomics流水线对来自同系肿瘤模型的CD45 +免疫细胞进行单细胞RNA测序（scRNA-seq）。我们发现CD47表达与CD68 + M的水平相关，但与CD163 + M2的水平无关。高水平的肿瘤浸润性CD68 + M，CD163 + M2和CD47表达与较差的生存率显着相关。CD47high / CD68 + Mhigh和CD47high / CD163 + M2high与较短的生存时间显着相关，而CD47low / CD68 + Mlow和CD47low / CD163 + M2low与较长的生存时间相关。有趣的是，CD47阻滞降低了Panc02中的肿瘤负担，但未降低MPC-83同系小鼠模型中的肿瘤负担。使用scRNA-seq，我们显示抗CD47处理可通过增加显示抗肿瘤功能的促炎巨噬细胞，同时减少抗炎巨噬细胞，显着重塑Panc02荷瘤小鼠的瘤内淋巴细胞和巨噬细胞区室。而且，CD47阻断不仅增加了肿瘤内CD8 + T细胞的数量，而且还可以将T细胞簇重塑为更活化的簇。此外，针对CD47和PD-L1的联合疗法在MPC-83中协同抑制了PDAC的生长，但在Panc02模型中却没有。用抗CD47和抗PD-L1处理的MPC-83小鼠但不包括Panc02小鼠，显示PD-1 + CD8 + T细胞数量增加，并且关键的免疫激活基因表达增强。我们的数据表明CD47靶向诱导PDAC中TME的肿瘤浸润免疫细胞的区室重塑。由于此组合治疗对不同PDAC细胞系建立的TME中浸润的免疫细胞和关键免疫激活基因的不同作用，因此不同的PDAC小鼠模型对抗CD47和抗PD-L1阻断表现出不同的反应。靶向CD47和PD-L1的联合疗法在MPC-83中协同抑制了PDAC的生长，但在Panc02模型中却没有。用抗CD47和抗PD-L1处理的MPC-83小鼠但不包括Panc02小鼠，显示PD-1 + CD8 + T细胞数量增加，并且关键的免疫激活基因表达增强。我们的数据表明CD47靶向诱导PDAC中TME的肿瘤浸润免疫细胞的区室重塑。由于此组合治疗对不同PDAC细胞系建立的TME中浸润的免疫细胞和关键免疫激活基因的不同作用，因此不同的PDAC小鼠模型对抗CD47和抗PD-L1阻断表现出不同的反应。靶向CD47和PD-L1的联合疗法在MPC-83中协同抑制了PDAC的生长，但在Panc02模型中却没有。用抗CD47和抗PD-L1处理的MPC-83小鼠但不包括Panc02小鼠，显示PD-1 + CD8 + T细胞数量增加，并且关键的免疫激活基因表达增强。我们的数据表明CD47靶向诱导PDAC中TME的肿瘤浸润免疫细胞的区室重塑。由于此组合治疗对不同PDAC细胞系建立的TME中浸润的免疫细胞和关键免疫激活基因的不同作用，因此不同的PDAC小鼠模型对抗CD47和抗PD-L1阻断表现出不同的反应。用抗CD47和抗PD-L1处理的MPC-83小鼠但不包括Panc02小鼠，显示PD-1 + CD8 + T细胞数量增加，并且关键的免疫激活基因表达增强。我们的数据表明CD47靶向诱导PDAC中TME的肿瘤浸润免疫细胞的区室重塑。由于此组合治疗对不同PDAC细胞系建立的TME中浸润的免疫细胞和关键免疫激活基因的不同作用，因此不同的PDAC小鼠模型对抗CD47和抗PD-L1阻断表现出不同的反应。用抗CD47和抗PD-L1处理的MPC-83小鼠但不包括Panc02小鼠，显示PD-1 + CD8 + T细胞数量增加，并且关键的免疫激活基因表达增强。我们的数据表明CD47靶向诱导PDAC中TME的肿瘤浸润免疫细胞的区室重塑。由于此组合治疗对不同PDAC细胞系建立的TME中浸润的免疫细胞和关键免疫激活基因的不同作用，因此不同的PDAC小鼠模型对抗CD47和抗PD-L1阻断表现出不同的反应。