Hepatic myofibroblasts exert immunosuppressive effects independent of the immune checkpoint regulator PD-L1 in liver metastasis of pancreatic ductal adenocarcinoma

Abstract

IntroductionPancreatic 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.MethodsFormalin-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.ResultsImmunohistochemical 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.ConclusionOur 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.

Epithelial and Mesenchymal-like Pancreatic Cancer Cells Exhibit Different Stem Cell Phenotypes Associated with Different Metastatic Propensities

“…These phenotypic switches of CSC populations, but also non-CSC populations (particularly Panc89 Paraclone tumors), and thus differing phenotypes from the in vitro analysis, could be explained by the exposure of the tumor cells to different microenvironmental niches. Thus, myofibroblasts and macrophages, both important stroma cell populations in pancreatic tumors and metastases [87,88], were already shown to induce L1CAM expression in PDAC cells [89][90][91] Altogether, these data indicate a robust phenotype stability of Holo-and Paraclones of either PDAC cell line under constant environmental conditions. Although, when the latter are changing, this might lead to a phenotypic switching of the tumor cells, presumably forced by the altered environmental factors the tumor cells are exposed to, as observed in the in vivo tumors.…”

The role of immunosuppressive myofibroblasts in the aging process and age-related diseases

“…This collaboration is emphasized in pathological conditions when the fibroblasts organize acute inflammatory responses and subsequently adapt to chronic inflammatory conditions. As discussed below, fibroblasts/myofibroblasts are key players in the generation and maintenance of immunosuppressive states, e.g., in the cancer microenvironment [ 6 , 75 , 76 ].…”

The challenge of making the right choice: patient avatars in the era of cancer immunotherapies

“…A further improvement of the above mentioned 2D cultures are spheroids which are three dimensional aggregates of one or multiple cell types. Spheroids can be comprised of tumor cells (primary cells or cell lines) only or of mixtures of tumor and stroma/immune cells ( 41 ). Furthermore, ECM can be supplemented.…”

“…Recently, 3D spheroids have been used to evaluate different immunotherapeutics, e.g. CAR NK cells against triple negative breast cancer ( 46 ), ICI targeting of PD-L1 in pancreatic ductal adenocarcinoma (PDAC) ( 41 ) or a strategy to activate tumor associated macrophages via CSF1R inhibition and CD40 activation in Her2-positive breast cancer ( 47 ).…”