IL-17A-producing CD8+ T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.

Electron Microscopy of Cells Grown on Polyacrylamide Hydrogels.

The composition of the cell culture environment profoundly affects cultured cells. Standard cell culture equipment such as plastic and glass provide extremely stiff surfaces compared to physiological cell environments (i.e., tissue). A growing body of evidence documents the artificial behavior and morphology of cells cultured on supraphysiologically stiff surfaces, such as glass (elastic modulus ca. 70,000 MPA) or plastic (e.g., polystyrol ca. 3300 MPA). Therefore, polymer-based hydrogels are increasingly employed as more physiologically appropriate (<100 kPA) supports for 2D or 3D culture. Since multiple properties that influence the cultured cells may be easily adjusted, hydrogels have become versatile tools for studying cells in a more native in vitro environment. Polyacrylamide-based hydrogels can be used as culture substrates for a broad variety of adherent cells and are easy to handle in most downstream biological assays, such as immunohistochemistry or molecular biology methods. We faced, however, serious difficulties with processing high stiffness polyacrylamide-based hydrogels for electron microscopy. To overcome this problem, we developed a simple protocol for embedding and processing cells grown on high stiffness polyacrylamide hydrogels that do not require modifications of routine embedding protocols. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Embedding of polyacrylamide-based hydrogels for transmission electron microscopy Alternate Protocol 1: Procedure for detached hydrogels Alternate Protocol 2: Procedure for attached hydrogels.

Matrix stiffness drives stromal autophagy and promotes formation of a protumorigenic niche.

Increased stiffness of solid tissues has long been recognized as a diagnostic feature of several pathologies, most notably malignant diseases. In fact, it is now well established that elevated tissue rigidity enhances disease progression and aggressiveness and is associated with a poor prognosis in patients as documented, for instance, for lung fibrosis or the highly desmoplastic cancer of the pancreas. The underlying mechanisms of the interplay between physical properties and cellular behavior are, however, not very well understood. Here, we have found that switching culture conditions from soft to stiff substrates is sufficient to evoke (macro) autophagy in various fibroblast types. Mechanistically, this is brought about by stiffness-sensing through an Integrin αV-focal adhesion kinase module resulting in sequestration and posttranslational stabilization of the metabolic master regulator AMPKα at focal adhesions, leading to the subsequent induction of autophagy. Importantly, stiffness-induced autophagy in stromal cells such as fibroblasts and stellate cells critically supports growth of adjacent cancer cells in vitro and in vivo. This process is Integrin αV dependent, opening possibilities for targeting tumor-stroma crosstalk. Our data thus reveal that the mere change in mechanical tissue properties is sufficient to metabolically reprogram stromal cell populations, generating a tumor-supportive metabolic niche.

Activation of Cilia-Independent Hedgehog/GLI1 Signaling as a Novel Concept for Neuroblastoma Therapy.

Although being rare in absolute numbers, neuroblastoma (NB) represents the most frequent solid tumor in infants and young children. Therapy options and prognosis are comparably good for NB patients except for the high risk stage 4 class. Particularly in adolescent patients with certain genetic alterations, 5-year survival rates can drop below 30%, necessitating the development of novel therapy approaches. The developmentally important Hedgehog (Hh) pathway is involved in neural crest differentiation, the cell type being causal in the etiology of NB. However, and in contrast to its function in some other cancer types, Hedgehog signaling and its transcription factor GLI1 exert tumor-suppressive functions in NB, rendering GLI1 an interesting new candidate for anti-NB therapy. Unfortunately, the therapeutic concept of pharmacological Hh/GLI1 pathway activation is difficult to implement as NB cells have lost primary cilia, essential organelles for Hh perception and activation. In order to bypass this bottleneck, we have identified a GLI1-activating small molecule which stimulates endogenous GLI1 production without the need for upstream Hh pathway elements such as Smoothened or primary cilia. This isoxazole compound potently abrogates NB cell proliferation and might serve as a starting point for the development of a novel class of NB-suppressive molecules.

The mammalian Hedgehog pathway is modulated by ANP32 proteins.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Transcriptional profiling has so far delineated four major MB subgroups of which one is driven by uncontrolled Hedgehog (Hh) signaling (SHH-MB). This pathway is amenable to drug targeting, yet clinically approved compounds exclusively target the transmembrane component Smoothened (SMO). Unfortunately, drug resistance against SMO inhibitors is encountered frequently, making the identification of novel Hh pathway components mandatory, which could serve as novel drug targets in the future. Here, we have used MB as a tool to delineate novel modulators of Hh signaling and have identified the Acidic Nuclear Phosphoprotein 32 (ANP32) family of proteins as novel regulators. The expression of all three family members (ANP32A, ANP32B, ANP32E) is increased in Hh-induced MB and their expression level is negatively associated with overall survival in SHH-MB patients. Mechanistically, we could find that ANP32 proteins function as positive modulators of mammalian Hh signaling upstream of GLI transcription factors. These findings add hitherto unknown regulators to the mammalian Hh signaling cascade and might spur future translational efforts to combat Hh-driven malignancies.

DYRK1B regulates Hedgehog-induced microtubule acetylation.

The posttranslational modification (PTM) of tubulin subunits is important for the physiological functions of the microtubule (MT) cytoskeleton. Although major advances have been made in the identification of enzymes carrying out MT-PTMs, little knowledge is available on how intercellular signaling molecules and their associated pathways regulate MT-PTM-dependent processes inside signal-receiving cells. Here we show that Hedgehog (Hh) signaling, a paradigmatic intercellular signaling system, affects the MT acetylation state in mammalian cells. Mechanistically, Hh pathway activity increases the levels of the MT-associated DYRK1B kinase, resulting in the inhibition of GSK3ß through phosphorylation of Serine 9 and the subsequent suppression of HDAC6 enzyme activity. Since HDAC6 represents a major tubulin deacetylase, its inhibition increases the levels of acetylated MTs. Through the activation of DYRK1B, Hh signaling facilitates MT-dependent processes such as intracellular mitochondrial transport, mesenchymal cell polarization or directed cell migration. Taken together, we provide evidence that intercellular communication through Hh signals can regulate the MT cytoskeleton and contribute to MT-dependent processes by affecting the level of tubulin acetylation.