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 Foxn1 ^nu/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 Foxn1 ^nu/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.

Significance: Increased tissue stiffness is a hallmark of several diseases such as organ fibrosis or cancer, and tissue fibroblasts play central roles in these pathologies. In tumors, stromal cells support the growth of cancer cells by supplying signaling molecules and nutrients, the latter also being provided by stromal autophagy. The mechanisms inducing these processes are, however, not well understood. Here, we demonstrate that an increased physical rigidity of a fibroblast’s environment is sufficient to evoke stromal autophagy. Mechanistically, matrix stiffness is transmitted via Integrin αV receptors and focal adhesion kinase, resulting in stabilization of AMPK at focal adhesion structures. Stiffness-mediated stromal autophagy supports the growth of neighboring tumor cells in vitro and in vivo, opening possibilities for anti-cancer therapies.

Tumor-associated macrophages (TAMs) play a pivotal role in the tumor microenvironment (TME), actively contributing to the formation of an immunosuppressive niche that fosters tumor progression. Consequently, there has been a growing interest in targeting TAMs as a promising avenue for cancer therapy. Recent advances in the field of immunometabolism have shed light on the influence of metabolic adaptations on macrophage physiology in the context of cancer. Here, we discuss the key metabolic pathways that shape the phenotypic diversity of macrophages. We place special emphasis on how metabolic reprogramming impacts the activation status of TAMs and their functions within the TME. Additionally, we explore alterations in TAM metabolism and their effects on phagocytosis, production of cytokines/chemokines and interaction with cytotoxic T and NK immune cells. Moreover, we examine the application of nanomedical approaches to target TAMs and assess the clinical significance of modulating the metabolism of TAMs as a strategy to develop new anti-cancer therapies. Taken together, in this comprehensive review article focusing on TAMs, we provide invaluable insights for the development of effective immunotherapeutic strategies and the enhancement of clinical outcomes for cancer patients.

Pancreatic cancer represents one of the most desmoplastic malignancies and is characterized by an extensive deposition of extracellular matrix. The latter is provided by activated cancer-associated fibroblasts (CAFs), which are abundant cells in the pancreatic tumor microenvironment. Many recent studies have made it clear that CAFs are not a singular cellular entity but represent a multitude of potentially dynamic subgroups that affect tumor biology at several levels. As mentioned before, CAFs significantly contribute to the fibrotic reaction and the biomechanical properties of the tumor, but they can also modulate the local immune environment and the response to targeted, chemo or radiotherapy. As the number of known and emerging CAF subgroups is steadily increasing, it is becoming increasingly difficult to keep up with these developments and to clearly discriminate the cellular subsets identified so far. This review aims to provide a helpful overview that enables readers to quickly familiarize themselves with field of CAF heterogeneity and to grasp the phenotypic, functional and therapeutic distinctions of the various stromal subpopulations.

Changes in cell number during in vitro experiments and pharmacological screenings primarily depend on two factors: cell death and proliferation. The dynamics of these processes determine whether cell populations expand and accumulate or, conversely, decrease over time. Understanding the biological mechanisms governing these changes is crucial for deciphering the mode of action of any pharmacological or genetic treatment in fundamental research and pre-clinical trials. In this context, we introduce a robust and efficient flow cytometry-based methodology that enables comprehensive analysis of key cellular parameters that indicate changes in cell numbers. This approach encompasses the assessment of cell count along with critical maintenance parameters including proliferation, cell cycle dynamics, apoptosis, cell permeability, and mitochondrial depolarization. These parameters are intricately linked, offering a detailed view of the cellular state. The described methodology is versatile and adaptable for analyzing various cell types, whether at steady state or in response to treatments. To develop this workflow, we integrated and optimised multiple flow cytometry-based stainings such as annexin V, propidium iodide, bromodeoxyuridine, CellTrace Violet, and JC-1 into a unified protocol. This article offers a detailed, step-by-step guide to the entire method, covering aspects such as timing, sample preparation techniques, and the reagents used. Additionally, it includes examples of the data that can be obtained with this technique and illustrates its multiparametric visualization. Collectively, this methodology facilitates the rapid acquisition of up to eight different parameters from a single sample in one experiment.