胰腺导管腺癌(PDAC)是西方国家癌症相关死亡的第四大常见原因。大多数病人被诊断为晚期,经常已经有转移。转移的主要部位是肝脏,肝肌成纤维细胞(HMF)在转移性生长中起关键作用。针对程序性死亡配体1(PD-L1)或程序性细胞死亡蛋白1(PD-1)的免疫检查点抑制剂(ICI)改善了几种癌症的治疗,但不是PDAC的治疗。因此,本研究旨在更好地了解HMF对肝转移过程中PDAC细胞PD-L1表达和免疫逃避的影响。
■使用福尔马林固定和石蜡包埋的活检标本或来自15例PDAC患者的肝转移的诊断性切除标本进行免疫组织化学分析。连续切片用针对泛细胞角蛋白的抗体染色,αSMA,CD8和PD-L1。探讨PD-1/PD-L1轴和HMF是否有助于PDAC肝转移的免疫逃逸,体外建立了富含基质的3D球体共培养模型,使用两种不同的PDAC细胞系,HMF,和CD8+T细胞。这里,进行了功能和流式细胞术分析。
■PDAC患者的肝组织切片的免疫组织化学分析显示HMF在肝转移中代表了丰富的基质群体,在小(1500µm)和大(>1500μm)转移的空间分布上有明显差异。在后者中,PD-L1表达主要位于侵袭前沿或均匀分布,而小的转移要么缺乏PD-L1表达,要么在中心表现出大部分弱表达。双重染色显示PD-L1主要由基质细胞表达,尤其是HMF。没有或低PD-L1表达的小肝转移在肿瘤中心包含更多的CD8+T细胞,而显示更强PD-L1表达的大转移包括较少的CD8+T细胞,主要位于侵袭前沿。富含HMF的球体共培养物,具有不同比例的PDAC细胞和HMF,可以很好地模拟原位肝转移的条件。这里,HMF损害了CD8+T细胞的效应分子的释放和PDAC细胞死亡的诱导,该效应取决于HMF的量以及PDAC细胞的量。ICI处理导致不同CD8+T细胞效应分子的分泌增加,但在任一球体条件下都不增加PDAC细胞死亡。
■我们的发现表明HMF的空间重组,CD8+T细胞,PDAC肝转移进展过程中PD-L1的表达。此外,HMF有效损害CD8+T细胞的效应子表型,但PD-L1/PD-1轴在这种情况下显然起次要作用,表明PDAC肝转移的免疫逃避依赖于其他免疫抑制机制。
UNASSIGNED: Pancreatic ductal adenocarcinoma (PDAC) represents the 4th most common cause of cancer-related deaths in Western countries. Most patients are diagnosed at advanced stages, often already with metastases. The main site of metastasis is the liver and hepatic myofibroblasts (HMF) play a pivotal role in metastatic outgrowth. Immune checkpoint inhibitors (ICI) targeting programmed death ligand 1 (PD-L1) or programmed cell death protein 1 (PD-1) improved treatment of several cancers but not of PDAC. Therefore, this study aimed to better understand the impact of HMF on PD-L1 expression and immune evasion of PDAC cells during liver metastasis.
UNASSIGNED: Formalin-fixed and paraffin embedded biopsy samples or diagnostic resection specimens from liver metastases of 15 PDAC patients were used for immunohistochemical analyses. Serial sections were stained with antibodies directed against Pan-Cytokeratin, αSMA, CD8, and PD-L1. To investigate whether the PD-1/PD-L1 axis and HMF contribute to immune escape of PDAC liver metastases, a stroma enriched 3D spheroid coculture model was established in vitro, using two different PDAC cell lines, HMF, and CD8+ T cells. Here, functional and flow cytometry analyses were conducted.
UNASSIGNED: Immunohistochemical analysis of liver tissue sections of PDAC patients revealed that HMF represent an abundant stroma population in liver metastases, with clear differences in the spatial distribution in small (1500 µm) and large (> 1500 μm) metastases. In the latter, PD-L1 expression was mainly located at the invasion front or evenly distributed, while small metastases either lacked PD-L1 expression or showed mostly weak expression in the center. Double stainings revealed that PD-L1 is predominantly expressed by stromal cells, especially HMF. Small liver metastases with no or low PD-L1 expression comprised more CD8+ T cells in the tumor center, while large metastases exhibiting stronger PD-L1 expression comprised less CD8+ T cells being mostly located at the invasion front. HMF-enriched spheroid cocultures with different ratios of PDAC cells and HMF well mimicking conditions of hepatic metastases in situ. Here, HMF impaired the release of effector molecules by CD8+ T cells and the induction of PDAC cell death, an effect that was dependent on the amount of HMF but also of PDAC cells. ICI treatment led to elevated secretion of distinct CD8+ T cell effector molecules but did not increase PDAC cell death under either spheroid condition.
UNASSIGNED: Our findings indicate a spatial reorganization of HMF, CD8+ T cells, and PD-L1 expression during progression of PDAC liver metastases. Furthermore, HMF potently impair the effector phenotype of CD8+ T cells but the PD-L1/PD-1 axis apparently plays a minor role in this scenario suggesting that immune evasion of PDAC liver metastases relies on other immunosuppressive mechanisms.