■免疫检查点抑制剂(ICI),例如,靶向程序性细胞死亡蛋白1-配体1(PD-L1)或其受体PD-1,已经显著改善了许多癌症的治疗,但迄今为止在胰腺导管腺癌(PDAC)中失败.巨噬细胞代表PDAC的肿瘤微环境(TME)中最丰富的免疫细胞群之一,能够根据其表型支持或抑制肿瘤进展。为了更好地了解PD-L1/PD-1抑制剂在PDAC中的治疗失败,这项研究在PDAC中动态TME的背景下检查了PD-L1的表达,特别关注巨噬细胞的影响。
■将原发性PDAC组织和相应的肝转移的福尔马林固定和石蜡包埋的组织样品用于免疫组织化学分析。连续切片用检测泛细胞角蛋白的抗体染色,CD68、CD163、CD8和PD-L1。为了研究PD-1/PD-L1轴和巨噬细胞是否有助于PDAC细胞的免疫逃逸,体外建立了富含基质的3D球体共培养模型,使用不同的PDAC细胞系和巨噬细胞亚型以及CD8+T细胞。进行功能和流式细胞术分析以表征细胞群体。
■免疫组织化学分析显示PD-L1主要由基质细胞表达,包括原发性PDAC组织和相应肝转移中的巨噬细胞而非PDAC细胞。值得注意的是,在CD8+T细胞和肿瘤细胞之间的肿瘤病变的侵袭前沿通常发现高局部巨噬细胞丰度和强PD-L1染色.为了研究表达PD-L1的巨噬细胞是否影响PDAC细胞对PD-L1/PD-1抑制剂治疗的反应,我们建立了一个球体模型,该模型包含两种不同的PDAC细胞系和不同比例的体外分化原代M1-或M2-样极化巨噬细胞.根据我们的现场发现,在巨噬细胞而不是PDAC细胞中观察到高PD-L1表达,PDAC细胞的存在进一步增加。PDAC巨噬细胞球状体增强了共培养的CD8+T细胞的效应表型,例如活化标志物的表达和效应分子的释放。特别是与M1样巨噬细胞相比单一培养球体。然而,这与PDAC细胞死亡增强无关.单独使用Durvalumab或Pembrolizumab或与吉西他滨组合的ICI治疗几乎不影响CD8+T细胞的效应表型以及PDAC细胞死亡。因此,尽管在巨噬细胞中有强烈的PD-L1表达,ICI处理没有导致CD8+T细胞的增强的活化和细胞毒性表型。
■总的来说,我们的研究揭示了在ICI存在下PDAC细胞和巨噬细胞相互作用的新见解.
Immune checkpoint inhibitors (ICI), e.g., targeting programmed cell death protein 1-ligand 1 (PD-L1) or its receptor PD-1, have markedly improved the therapy of many cancers but so far failed in pancreatic ductal adenocarcinoma (PDAC). Macrophages represent one of the most abundant immune cell populations within the tumor microenvironment (TME) of PDAC being able to either support or restrain tumor progression depending on their phenotype. To better understand treatment failure of PD-L1/PD-1 inhibitors in PDAC, this study examined PD-L1 expression in the context of a dynamic TME in PDAC with a particular focus on the impact of macrophages.
Formalin-fixed and paraffin embedded tissue samples of primary PDAC tissues and corresponding liver metastases were used for immunohistochemical analyses. Serial sections were stained with antibodies detecting Pan-Cytokeratin, CD68, CD163, CD8, and PD-L1.To investigate whether the PD-1/PD-L1 axis and macrophages contribute to immune escape of PDAC cells, a stroma enriched 3D spheroid coculture model was established in vitro, using different PDAC cell lines and macrophages subtypes as well as CD8+ T cells. Functional and flow cytometry analyses were conducted to characterize cell populations.
Immunohistochemical analyses revealed that PD-L1 is mainly expressed by stroma cells, including macrophages and not PDAC cells in primary PDAC tissues and corresponding liver metastases. Notably, high local abundance of macrophages and strong PD-L1 staining were commonly found at invasion fronts of tumoral lesions between CD8+ T cells and tumor cells. In order to investigate whether PD-L1 expressing macrophages impact the response of PDAC cells to treatment with PD-L1/PD-1 inhibitors, we developed a spheroid model comprising two different PDAC cell lines and different ratios of in vitro differentiated primary M1- or M2-like polarized macrophages. In line with our in situ findings, high PD-L1 expression was observed in macrophages rather than PDAC cells, which was further increased by the presence of PDAC cells. The effector phenotype of co-cultured CD8+ T cells exemplified by expression of activation markers and release of effector molecules was rather enhanced by PDAC macrophage spheroids, particularly with M1-like macrophages compared to mono-culture spheroids. However, this was not associated with enhanced PDAC cell death. ICI treatment with either Durvalumab or Pembrolizumab alone or in combination with Gemcitabine hardly affected the effector phenotype of CD8+ T cells along with PDAC cell death. Thus, despite strong PD-L1 expression in macrophages, ICI treatment did not result in an enhanced activation and cytotoxic phenotype of CD8+ T cells.
Overall, our study revealed novel insights into the interplay of PDAC cells and macrophages in the presence of ICI.