Stiffness-induced cancer-associated fibroblasts are responsible for immunosuppression in a platelet-derived growth factor ligand-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a vast stromal reaction that arises mainly from cancer-associated fibroblasts (CAFs) and promotes both immune escape and tumor growth. Here, we used a mouse model with deletion of the activin A receptor ALK4 in the context of the KrasG12D mutation, which strongly drives collagen deposition that leads to tissue stiffness. By ligand-receptor analysis of single-cell RNA-sequencing data, we identified that, in stiff conditions, neoplastic ductal cells instructed CAFs through sustained platelet-derived growth factor (PDGF) signaling. Tumor-associated tissue rigidity resulted in the emergence of stiffness-induced CAFs (siCAFs) in vitro and in vivo. Similar results were confirmed in human data. siCAFs were able to strongly inhibit CD8+ T-cell responses in vitro and in vivo, promoting local immunosuppression. More importantly, targeting PDGF signaling led to diminished siCAF and reduced tumor growth. Our data show for the first time that early paracrine signaling leads to profound changes in tissue mechanics, impacting immune responses and tumor progression. Our study highlights that PDGF ligand neutralization can normalize the tissue architecture independent of the genetic background, indicating that finely tuned stromal therapy may open new therapeutic avenues in pancreatic cancer.

Proteomics of Plasma and Plasma-Treated Podocytes: Application to Focal and Segmental Glomerulosclerosis.

Focal and segmental glomerulosclerosis (FSGS) is a severe form of idiopathic nephrotic syndrome (INS), a glomerulopathy of presumably immune origin that is attributed to extrarenal pathogenic circulating factors. The recurrence of FSGS (rFSGS) after transplant occurs in 30% to 50% of cases. The direct analysis of patient plasma proteome has scarcely been addressed to date, mainly due to the methodological difficulties associated with plasma complexity and dynamic range. In this study, first, we compared different methods of plasma preparation, second, we compared the plasma proteomes of rFSGS and controls using two preparation methods, and third, we analyzed the early proximal signaling events in podocytes subjected to patient plasma, through a combination of phosphoproteomics and lipid-raft proteomics (raftomics). By combining immunodepletion and high pH fractionation, we performed a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) obtained from healthy controls, non-INS patient controls, and rFSGS patients (n = 4). In both the soluble- and the EV-protein sets from the rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid-binding proteins, generally associated with lipoproteins, was found to be decreased in the soluble set from the rFSGS patients. In addition, three amino acid transporters involved in mTORC1 activation were found to be significantly increased in the EV from the rFSGS. Next, we incubated human podocytes for 30 min with 10% plasma from both groups of patients. The phosphoproteomics and raftomics of the podocytes revealed profound differences in the proteins involved in the mTOR pathway, in autophagy, and in cytoskeleton organization. We analyzed the correlation between the abundance of plasma and plasma-regulated podocyte proteins. The observed changes highlight some of the mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating-factor activity in podocytes.

Gangliosides in Podocyte Biology and Disease.

Gangliosides constitute a subgroup of glycosphingolipids characterized by the presence of sialic acid residues in their structure. As constituents of cellular membranes, in particular of raft microdomains, they exert multiple functions, some of them capital in cell homeostasis. Their presence in cells is tightly regulated by a balanced expression and function of the enzymes responsible for their biosynthesis, ganglioside synthases, and their degradation, glycosidases. The dysregulation of their abundance results in rare and common diseases. In this review, we make a point on the relevance of gangliosides and some of their metabolic precursors, such as ceramides, in the function of podocytes, the main cellular component of the glomerular filtration barrier, as well as their implications in podocytopathies. The results presented in this review suggest the pertinence of clinical lipidomic studies targeting these metabolites.

CMIP is a negative regulator of T cell signaling.

Upon their interaction with cognate antigen, T cells integrate different extracellular and intracellular signals involving basal and induced protein-protein interactions, as well as the binding of proteins to lipids, which can lead to either cell activation or inhibition. Here, we show that the selective T cell expression of CMIP, a new adapter protein, by targeted transgenesis drives T cells toward a naïve phenotype. We found that CMIP inhibits activation of the Src kinases Fyn and Lck after CD3/CD28 costimulation and the subsequent localization of Fyn and Lck to LRs. Video microscopy analysis showed that CMIP blocks the recruitment of LAT and the lipid raft marker cholera toxin B at the site of TCR engagement. Proteomic analysis identified several protein clusters differentially modulated by CMIP and, notably, Cofilin-1, which is inactivated in CMIP-expressing T cells. Moreover, transgenic T cells exhibited the downregulation of GM3 synthase, a key enzyme involved in the biosynthesis of gangliosides. These results suggest that CMIP negatively impacts proximal signaling and cytoskeletal rearrangement and defines a new mechanism for the negative regulation of T cells that could be a therapeutic target.