Expression and role of the immune checkpoint regulator PD-L1 in the tumor-stroma interplay of pancreatic ductal adenocarcinoma.

Introduction: 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. Methods: 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. Results: 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. Conclusion: Overall, our study revealed novel insights into the interplay of PDAC cells and macrophages in the presence of ICI.

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

Introduction: 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. Methods: 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. Results: 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. Conclusion: 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.

Meprin and ADAM proteases as triggers of systemic inflammation in sepsis.

Systemic inflammatory disorders (SIDs) comprise a broad range of diseases characterized by dysregulated excessive innate immune responses. Severe forms of SIDs can lead to organ failure and death, and their increasing incidence represents a major issue for the healthcare system. Protease-mediated ectodomain shedding of cytokines and their receptors represents a central mechanism in the regulation of inflammatory responses. The metalloprotease A disintegrin and metalloproteinase (ADAM) 17 is the best-characterized ectodomain sheddase capable of releasing TNF-α and soluble IL-6 receptor, which are decisive factors of systemic inflammation. Recently, meprin metalloproteases were also identified as IL-6 receptor sheddases and activators of the pro-inflammatory cytokines IL-1ß and IL-18. In different mouse models of SID, particularly those mimicking a sepsis-like phenotype, ADAM17 and meprins have been found to promote disease progression. In this review, we summarize the role of ADAM10, ADAM17, and meprins in the onset and progression of sepsis and discuss their potential as therapeutic targets.

Syndecan-1 shedding by meprin ß impairs keratinocyte adhesion and differentiation in hyperkeratosis.

Dysregulation of proteolytic enzymes has huge impact on epidermal homeostasis, which can result in severe pathological conditions such as fibrosis or Netherton syndrome. The metalloprotease meprin ß was found to be upregulated in hyperproliferative skin diseases. AP-1 transcription factor complex has been reported to induce Mep1b expression. Since AP-1 and its subunit fos-related antigen 2 (fra-2) are associated with the onset and progression of psoriasis, we wanted to investigate if this could partially be attributed to increased meprin ß activity. Here, we demonstrate that fra-2 transgenic mice show increased meprin ß expression and proteolytic activity in the epidermis. To avoid influence by other fra-2 regulated genes, we additionally generated a mouse model that enabled tamoxifen-inducible expression of meprin ß under the Krt5-promotor to mimic the pathological condition. Interestingly, induced meprin ß expression in the epidermis resulted in hyperkeratosis, hair loss and mottled pigmentation of the skin. Employing N-terminomics revealed syndecan-1 as a substrate of meprin ß in skin. Shedding of syndecan-1 at the cell surface caused delayed calcium-induced differentiation and impaired adhesion of keratinocytes, which was blocked by the meprin ß inhibitor fetuin-B.

Initiation of Pancreatic Cancer: The Interplay of Hyperglycemia and Macrophages Promotes the Acquisition of Malignancy-Associated Properties in Pancreatic Ductal Epithelial Cells.

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive solid malignancies with a poor prognosis. Obesity and type 2 diabetes mellitus (T2DM) are two major risk factors linked to the development and progression of PDAC, both often characterized by high blood glucose levels. Macrophages represent the main immune cell population in PDAC contributing to PDAC development. It has already been shown that pancreatic ductal epithelial cells (PDEC) undergo epithelial-mesenchymal transition (EMT) when exposed to hyperglycemia or macrophages. Thus, this study aimed to investigate whether concomitant exposure to hyperglycemia and macrophages aggravates EMT-associated alterations in PDEC. Exposure to macrophages and elevated glucose levels (25 mM glucose) impacted gene expression of EMT inducers such as IL-6 and TNF-α as well as EMT transcription factors in benign (H6c7-pBp) and premalignant (H6c7-kras) PDEC. Most strikingly, exposure to hyperglycemic coculture with macrophages promoted downregulation of the epithelial marker E-cadherin, which was associated with an elevated migratory potential of PDEC. While blocking IL-6 activity by tocilizumab only partially reverted the EMT phenotype in H6c7-kras cells, neutralization of TNF-α by etanercept was able to clearly impair EMT-associated properties in premalignant PDEC. Altogether, the current study attributes a role to a T2DM-related hyperglycemic, inflammatory micromilieu in the acquisition of malignancy-associated alterations in premalignant PDEC, thus providing new insights on how metabolic diseases might promote PDAC initiation.

Chitosan nanoparticles as antigen vehicles to induce effective tumor specific T cell responses.

Cancer vaccinations sensitize the immune system to recognize tumor-specific antigens de novo or boosting preexisting immune responses. Dendritic cells (DCs) are regarded as the most potent antigen presenting cells (APCs) for induction of (cancer) antigen-specific CD8+ T cell responses. Chitosan nanoparticles (CNPs) used as delivery vehicle have been shown to improve anti-tumor responses. This study aimed at exploring the potential of CNPs as antigen delivery system by assessing activation and expansion of antigen-specific CD8+ T cells by DCs and subsequent T cell-mediated lysis of pancreatic ductal adenocarcinoma (PDAC) cells. As model antigen the ovalbumin-derived peptide SIINFEKL was chosen. Using imaging cytometry, intracellular uptake of FITC-labelled CNPs of three different sizes and qualities (90/10, 90/20 and 90/50) was demonstrated in DCs and in pro- and anti-inflammatory macrophages to different extents. While larger particles (90/50) impaired survival of all APCs, small CNPs (90/10) were not toxic for DCs. Internalization of SIINFEKL-loaded but not empty 90/10-CNPs promoted a pro-inflammatory phenotype of DCs indicated by elevated expression of pro-inflammatory cytokines. Treatment of murine DC2.4 cells with SIINFEKL-loaded 90/10-CNPs led to a marked MHC-related presentation of SIINFEKL and enabled DC2.4 cells to potently activate SIINFEKL-specific CD8+ OT-1 T cells finally leading to effective lysis of the PDAC cell line Panc-OVA. Overall, our study supports the suitability of CNPs as antigen vehicle to induce potent anti-tumor immune responses by activation and expansion of tumor antigen-specific CD8+ T cells.

Inflammation Associated Pancreatic Tumorigenesis: Upregulation of Succinate Dehydrogenase (Subunit B) Reduces Cell Growth of Pancreatic Ductal Epithelial Cells.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most fatal malignancies and its development is highly associated with inflammatory processes such as chronic pancreatitis (CP). Since the succinate dehydrogenase subunit B (SDHB) is regarded as tumor suppressor that is lost during cancer development, this study investigated the impact of M1-macrophages as part of the inflammatory microenvironment on the expression as well as function of SDHB in benign and premalignant pancreatic ductal epithelial cells (PDECs). Immunohistochemical analyses on pancreatic tissue sections from CP patients and control individuals revealed a stronger SDHB expression in ducts of CP tissues being associated with a greater abundance of macrophages compared to ducts in control tissues. Accordingly, indirect co-culture with M1-macrophages led to clearly elevated SDHB expression and SDH activity in benign H6c7-pBp and premalignant H6c7-kras PDECs. While siRNA-mediated SDHB knockdown in these cells did not affect glucose and lactate uptake after co-culture, SDHB knockdown significantly promoted PDEC growth which was associated with increased proliferation and decreased effector caspase activity particularly in co-cultured PDECs. Overall, these data indicate that SDHB expression and SDH activity are increased in PDECs when exposed to pro-inflammatory macrophages as a counterregulatory mechanism to prevent excessive PDEC growth triggered by the inflammatory environment.

The Hepatic Microenvironment and TRAIL-R2 Impact Outgrowth of Liver Metastases in Pancreatic Cancer after Surgical Resection.

Most patients with pancreatic ductal adenocarcinoma (PDAC) undergoing curative resection relapse within months, often with liver metastases. The hepatic microenvironment determines induction and reversal of dormancy during metastasis. Both tumor growth and metastasis depend on the Tumor necrosis factor (TNF)-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2). This study investigated the interplay of TRAIL-R2 and the hepatic microenvironment in liver metastases formation and the impact of surgical resection. Although TRAIL-R2-knockdown (PancTu-I shTR2) decreased local relapses and number of macroscopic liver metastases after primary tumor resection in an orthotopic PDAC model, the number of micrometastases was increased. Moreover, abdominal surgery induced liver inflammation involving activation of hepatic stellate cells (HSCs) into hepatic myofibroblasts (HMFs). In coculture with HSCs, proliferation of PancTu-I shTR2 cells was significantly lower compared to PancTu-I shCtrl cells, an effect still observed after switching coculture from HSC to HMF, mimicking surgery-mediated liver inflammation and enhancing cell proliferation. CXCL-8/IL-8 blockade diminished HSC-mediated growth inhibition in PancTu-I shTR2 cells, while Vascular Endothelial Growth Factor (VEGF) neutralization decreased HMF-mediated proliferation. Overall, this study points to an important role of TRAIL-R2 in PDAC cells in the interplay with the hepatic microenvironment during metastasis. Resection of primary PDAC seems to induce liver inflammation, which might contribute to outgrowth of liver metastases.

Metastasis of pancreatic cancer: An uninflamed liver micromilieu controls cell growth and cancer stem cell properties by oxidative phosphorylation in pancreatic ductal epithelial cells.

Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed when liver metastases already emerged. We recently demonstrated that hepatic stromal cells determine the dormancy status along with cancer stem cell (CSC) properties of pancreatic ductal epithelial cells (PDECs) during metastasis. This study investigated the influence of the hepatic microenvironment - and its inflammatory status - on metabolic alterations and how these impact cell growth and CSC-characteristics of PDECs. Coculture with hepatic stellate cells (HSCs), simulating a physiological liver stroma, but not with hepatic myofibroblasts (HMFs) representing liver inflammation promoted expression of Succinate Dehydrogenase subunit B (SDHB) and an oxidative metabolism along with a quiescent phenotype in PDECs. SiRNA-mediated SDHB knockdown increased cell growth and CSC-properties. Moreover, liver micrometastases of tumor bearing KPC mice strongly expressed SDHB while expression of the CSC-marker Nestin was exclusively found in macrometastases. Consistently, RNA-sequencing and in silico modeling revealed significantly altered metabolic fluxes and enhanced SDH activity predominantly in premalignant PDECs in the presence of HSC compared to HMF. Overall, these data emphasize that the hepatic microenvironment determines the metabolism of disseminated PDECs thereby controlling cell growth and CSC-properties during liver metastasis.

Impact of p53 status on TRAIL-mediated apoptotic and non-apoptotic signaling in cancer cells.

Due to their ability to preferentially induce cell death in tumor cells, while sparing healthy cells, TNF-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL-R1 or anti-TRAIL-R2-specific antibodies are under clinical investigations for cancer-treatment. However, TRAIL-Rs may also induce signaling pathways, which result in malignant progression. TRAIL receptors are transcriptionally upregulated via wild-type p53 following radio- or chemotherapy. Nevertheless, the impact of p53 status on the expression and signaling of TRAIL-Rs is not fully understood. Therefore, we analyzed side by side apoptotic and non-apoptotic signaling induced by TRAIL or the agonistic TRAIL-R-specific antibodies Mapatumumab (anti-TRAIL-R1) and Lexatumumab (anti-TRAIL-R2) in the two isogenic colon carcinoma cell lines HCT116 p53+/+ and p53-/-. We found that HCT116 p53+/+ cells were significantly more sensitive to TRAIL-R-triggering than p53-/- cells. Similarly, A549 lung cancer cells expressing wild-type p53 were more sensitive to TRAIL-R-mediated cell death than their derivatives with knockdown of p53. Our data demonstrate that the contribution of p53 in regulating TRAIL-R-induced apoptosis does not correlate to the levels of TRAIL-Rs at the plasma membrane, but rather to p53-mediated upregulation of Bax, favouring the mitochondrial amplification loop. Consistently, stronger caspase-9 and caspase-3 activation as well as PARP-cleavage was observed following TRAIL-R-triggering in HCT116 p53+/+ compared to HCT116 p53-/- cells. Interestingly, HCT116 p53+/+ cells showed also a more potent activation of non-canonical TRAIL-R-induced signal transduction pathways like JNK, p38 and ERK1/ERK2 than p53-/- cells. Likewise, these cells induced IL-8 expression in response to TRAIL, Mapatumumab or Lexatumumab significantly stronger than p53-/- cells. We obtained similar results in A549 cells with or without p53-knockdown and in the two isogenic colon cancer cell lines RKO p53+/+ and p53-/-. In both cellular systems, we could clearly demonstrate the potentiating effects of p53 on TRAIL-R-mediated IL-8 induction. In conclusion, we found that wild-type p53 increases TRAIL-R-mediated apoptosis but simultaneously augments non-apoptotic signaling.

POLE Score: a comprehensive profiling of programmed death 1 ligand 1 expression in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) being characterized by a pronounced stromal compartment is commonly diagnosed at an advanced stage limiting curative treatment options. Although therapeutical targeting of immune checkpoint regulators like programmed death 1 ligand 1 (PD-L1) represent a promising approach that substantially improved survival of several highly aggressive malignancies, convincing indicators for response prediction are still lacking for PDAC which might be attributed to the insufficient characterization of PD-L1 status. Therefore, we investigated PD-L1 expression by immunohistochemistry in a well characterized cohort of 59 PDAC and 18 peritumoral tissues. Despite the histopathological homogeneity within our cohort, tumor tissues exhibited a great heterogeneity regarding PD-L1 expression. Considering distinct PD-L1 expression patterns, we established the novel POLE Score that incorporates overall PD-L1 expression (P), cellular Origin of PD-L1 (O), PD-L1 level in tumor-associated Lymph follicles (L) and Enumerated local PD-L1 distribution (E). We show that tumoral PD-L1 expression is higher compared to peritumoral areas. Furthermore, POLE Score parameters correlated with overall survival, tumor grade, Ki67 status, local proximity of tumor cells and particular stroma composition. For the first time, we demonstrate that PD-L1 is mostly expressed by stroma and rarely by tumor cells in PDAC. Moreover, our in situ analyses on serial tissue sections and in vitro data suggest that PD-L1 is prominently expressed by tumor-associated macrophages. In conclusion, POLE Score represents a comprehensive characterization of PD-L1 expression in tumor and stroma compartment and might provide the basis for improved patient stratification in future clinical trials on PD-1/PD-L1 targeting therapies in PDAC.

Liver metastasis of pancreatic cancer: the hepatic microenvironment impacts differentiation and self-renewal capacity of pancreatic ductal epithelial cells.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced stages with the liver as the main site of metastases. The hepatic microenvironment has been shown to determine outgrowth of liver metastases. Cancer stem cells (CSCs) are essential for initiation and maintenance of tumors and acquisition of CSC-properties has been linked to Epithelial-Mesenchymal-Transition. Thus, this study aimed at elucidating whether and how the hepatic microenvironment impacts stemness and differentiation of disseminated pancreatic ductal epithelial cells (PDECs). Culture of premalignant H6c7-kras and malignant Panc1 PDECs together with hepatocytes and hepatic stellate cells (HSC) promoted self-renewal capacity of both PDEC lines. This was indicated by higher colony formation compared to cells cocultured with hepatocytes and hepatic myofibroblasts. Different Panc1 colony types derived from an HSC-enriched coculture were expanded and characterized revealing that holoclones exhibited an enhanced colony formation ability, elevated and exclusive expression of the CSC-marker Nestin and a more pronounced mesenchymal phenotype compared to paraclones. Moreover, Panc1 holoclone cells showed an increased tumorigenic potential in vivo leading to formation of undifferentiated tumors in 7/10 animals, while inoculation of paraclone cells only led to formation of tumors in 2/10 animals being smaller in number and size. Holoclone tumors were characterized by elevated expression of mesenchymal markers, complete loss of E-cadherin expression and high expression of Nestin. Finally, Etanercept-mediated TNF-α blocking partly reversed the mesenchymal CSC-phenotype of Panc1 holoclone cells. Overall, these data provide evidence that the hepatic microenvironment determines stemness and differentiation of PDECs, thereby substantially contributing to liver metastases of PDAC.

Diabetes as risk factor for pancreatic cancer: Hyperglycemia promotes epithelial-mesenchymal-transition and stem cell properties in pancreatic ductal epithelial cells.

Type 2 diabetes mellitus (T2DM) is associated with hyperglycemia and a risk to develop pancreatic ductal adenocarcinoma (PDAC), one of the most fatal malignancies. Cancer stem cells (CSC) are essential for initiation and maintenance of tumors, and acquisition of CSC-features is linked to epithelial-mesenchymal-transition (EMT). The present study investigated whether hyperglycemia promotes EMT and CSC-features in premalignant and malignant pancreatic ductal epithelial cells (PDEC). Under normoglycemia (5 mM d-glucose), Panc1 PDAC cells but not premalignant H6c7-kras cells exhibited a mesenchymal phenotype along with pronounced colony formation. While hyperglycemia (25 mM d-glucose) did not impact the mesenchymal phenotype of Panc1 cells, CSC-properties were aggravated exemplified by increased Nanog expression and Nanog-dependent formation of holo- and meroclones. In H6c7-kras cells, high glucose increased secretion of Transforming-Growth-Factor-beta1 (TGF-ß1) as well as TGF-ß1 signaling, and in a TGF-ß1-dependent manner reduced E-cadherin expression, increased Nestin expression and number of meroclones. Finally, reduced E-cadherin expression was detected in pancreatic ducts of hyperglycemic but not normoglycemic mice. These data suggest that hyperglycemia promotes the acquisition of mesenchymal and CSC-properties in PDEC by activating TGF-ß signaling and might explain how T2DM facilitates pancreatic tumorigenesis.