Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells.

Glioblastoma (GBM) is a poorly treatable disease due to the fast development of tumor recurrences and high resistance to chemo- and radiotherapy. To overcome the highly adaptive behavior of GBMs, especially multimodal therapeutic approaches also including natural adjuvants have been investigated. However, despite increased efficiency, some GBM cells are still able to survive these advanced treatment regimens. Given this, the present study evaluates representative chemoresistance mechanisms of surviving human GBM primary cells in a complex in vitro co-culture model upon sequential application of temozolomide (TMZ) combined with AT101, the R(-) enantiomer of the naturally occurring cottonseed-derived gossypol. Treatment with TMZ+AT101/AT101, although highly efficient, yielded a predominance of phosphatidylserine-positive GBM cells over time. Analysis of the intracellular effects revealed phosphorylation of AKT, mTOR, and GSK3ß, resulting in the induction of various pro-tumorigenic genes in surviving GBM cells. A Torin2-mediated mTOR inhibition combined with TMZ+AT101/AT101 partly counteracted the observed TMZ+AT101/AT101-associated effects. Interestingly, treatment with TMZ+AT101/AT101 concomitantly changed the amount and composition of extracellular vesicles released from surviving GBM cells. Taken together, our analyses revealed that even when chemotherapeutic agents with different effector mechanisms are combined, a variety of chemoresistance mechanisms of surviving GBM cells must be taken into account.

Interleukin-6 controls recycling and degradation, but not internalization of its receptors.

Interleukin-6 (IL-6) is a cytokine implicated in proinflammatory as well as regenerative processes and acts via receptor complexes consisting of the ubiquitously expressed, signal-transducing receptor gp130 and the IL-6 receptor (IL-6R). The IL-6R is expressed only on hepatocytes and subsets of leukocytes, where it mediates specificity of the receptor complex to IL-6 as the subunit gp130 is shared with all other members of the IL-6 cytokine family such as IL-11 or IL-27. The amount of IL-6R at the cell surface thus determines the responsiveness of the cell to the cytokine and might therefore be decisive in the development of inflammatory disorders. However, how the expression levels of IL-6R and gp130 at the cell surface are controlled is largely unknown. Here, we show that IL-6R and gp130 are constitutively internalized independent of IL-6. This process depends on dynamin and clathrin and is temporally controlled by motifs within the intracellular region of gp130 and IL-6R. IL-6 binding and internalization of the receptors is a prerequisite for activation of the Jak/STAT signaling cascade. Targeting of gp130, but not of the IL-6R, to the lysosome for degradation depends on stimulation with IL-6. Furthermore, we show that after internalization and activation of signaling, both the IL-6R and gp130 are recycled back to the cell surface, a process that is enhanced by IL-6. These data reveal an important function of IL-6 beyond the pure activation of signaling.

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.

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.

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.

Biomarkers in Liquid Biopsies for Prediction of Early Liver Metastases in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies with poor survival rates. Only 20% of the patients are eligible for R0-surgical resection, presenting with early relapses, mainly in the liver. PDAC patients with hepatic metastases have a worse outcome compared to patients with metastases at other sites. Early detection of hepatic spread bears the potential to improve patient outcomes. Thus, this study sought for serum-based perioperative biomarkers allowing discrimination of early (EHMS ≤ 12 months) and late hepatic metastatic spread (LHMS > 12 months). Serum samples from 83 resectable PDAC patients were divided into EHMS and LHMS and analyzed for levels of inflammatory mediators by LEGENDplexTM, which was validated and extended by Olink® analysis. CA19-9 serum levels served as control. Results were correlated with clinicopathological data. While serum CA19-9 levels were comparable, Olink® analysis confirmed distinct differences between both groups. It revealed significantly elevated levels of factors involved in chemotaxis and migration of immune cells, immune activity, and cell growth in serum of LHMS-patients. Overall, Olink® analysis identified a comprehensive biomarker panel in serum of PDAC patients that could provide the basis for predicting LHMS. However, further studies with larger cohorts are required for its clinical translation.

Programmed Death-Ligand 1 (PD-L1) Expression Is Induced by Insulin in Pancreatic Ductal Adenocarcinoma Cells Pointing to Its Role in Immune Checkpoint Control.

Type-2 diabetes (T2DM) is a risk factor for the development of pancreatic ductal adenocarcinoma (PDAC) and is characterized by insulin resistance and hyperinsulinemia. Besides the well-known growth-promoting activity of insulin or the other members of the Insulin/Insulin-like Growth factor (IGF) axis, we here describe an inducing effect of insulin on PD-L1 expression in PDAC cells. Treatment of the PDAC cell lines BxPc3, A818-6, and T3M4 with insulin increased PD-L1 expression in a time- and dose dependent fashion, as shown by Western blot and qPCR analysis. siRNA mediated knock-down showed that the effects of insulin on PD-L1 depend on the insulin and IGF receptors (InsR and IGFR, respectively). In addition, a crosstalk of insulin-induced ERK activation and Epidermal Growth Factor (EGF) triggered PD-L1 expression. This involves different mechanisms in the three cell lines including upregulation of InsR-A expression in A818-6 and modulation of the adaptor protein Gab1 in BxPc3 cells. As a consequence of the insulin-induced PD-L1 expression, PDAC cells suppress the proliferation of activated human CD8+ T-cells in coculture experiments. The suppression of CD8+ cell proliferation by insulin-pretreated PDAC cells was reversed by PD-1 blockade with Pembrolizumab or by PD-L1 siRNA. Furthermore, the clinical relevance of these observations was supported by detecting a coexpression of cytoplasmic InsR (characteristic for its activation) and PD-L1 in tumor tissues from PDAC patients. Our findings provide a novel insight into the protumorigenic role of insulin in PDAC. Recognizing the impact of insulin on PD-L1 expression as part of the immune privilege, strategies to interfere with this mechanism could pave the way towards a more efficient immunotherapy of PDAC.