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Pancreatic ductal adenocarcinoma (PDA) has a highly immunosuppressive microenvironment, which is contributed by the complex interaction between cancer cells and a heterogeneous population of stromal cells. Therefore, facile and trackable models are needed for integrative and dynamic interrogation of cancer-stroma interaction. Here, we tracked the immunoevolution of PDA in a genetically-defined transplantable model of mouse pancreatic tumour organoids that recapitulates the progression of the disease from early preinvasive lesions to metastatic carcinomas. We demonstrated that organoid-derived isografts (ODI) can be used as a biological source of biomarkers (NT5E, TGFB1, FN1, and ITGA5) of aggressive molecular subtypes of human PDA. In ODI, infiltration from leukocytes is an early event during progression of the disease as observed for autochthonous models. Neoplastic progression was associated to accumulation of Maf+ macrophages, which inversely correlated with CD8+ T cells infiltration. Consistently, levels of MAF were enriched in human PDA subtypes characterized by abundance of macrophage-related transcripts and indicated poor patients’ survival. Density of MAF+ macrophages was higher in human PDA tissues compared to preinvasive lesions. Our results suggest that ODIs represent a suitable system for genotypic-immunophenotypic studies and support the hypothesis of MAF+ macrophages as a prominent immunosuppressive population in PDA.

Peroxiredoxin-2 (Prx2), a typical two-cysteine peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2(-/-) mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2(-/-) red cells and reduced Prx2(-/-) red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2(-/-) mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2(-/-) mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2(-/-) mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.

Chorea-acanthocytosis is one of the hereditary neurodegenerative disorders known as the neuroacanthocytoses. Chorea-acanthocytosis is characterized by circulating acanthocytes deficient in chorein, a protein of unknown function. We report here for the first time that chorea-acanthocytosis red cells are characterized by impaired autophagy, with cytoplasmic accumulation of active Lyn and of autophagy-related proteins Ulk1 and Atg7. In chorea-acanthocytosis erythrocytes, active Lyn is sequestered by HSP90-70 to form high-molecular-weight complexes that stabilize and protect Lyn from its proteasomal degradation, contributing to toxic Lyn accumulation. An interplay between accumulation of active Lyn and autophagy was found in chorea-acanthocytosis based on Lyn coimmunoprecipitation with Ulk1 and Atg7 and on the presence of Ulk1 in Lyn-containing high-molecular-weight complexes. In addition, chorein associated with Atg7 in healthy but not in chorea-acanthocytosis erythrocytes. Electron microscopy detected multivesicular bodies and membrane remnants only in circulating chorea-acanthocytosis red cells. In addition, reticulocyte-enriched chorea-acanthocytosis red cell fractions exhibited delayed clearance of mitochondria and lysosomes, further supporting the impairment of authophagic flux. Because autophagy is also important in erythropoiesis, we studied in vitro CD34+-derived erythroid precursors. In chorea-acanthocytosis, we found (1) dyserythropoiesis; (2) increased active Lyn; (3) accumulation of a marker of autophagic flux and autolysososme degradation; (4) accumlation of Lamp1, a lysosmal membrane protein, and LAMP1-positive aggregates; and (5) reduced clearance of lysosomes and mitochondria. Our results uncover in chorea-acanthocytosis erythroid cells an association between accumulation of active Lyn and impaired autophagy, suggesting a link between chorein and autophagic vesicle trafficking in erythroid maturation.

Chorea-acanthocytosis (ChAc) is a fatal neurological disorder characterized by red blood cell acanthocytes and striatal neurodegeneration. Recently, severe cell membrane disturbances based on depolymerized cortical actin and an elevated Lyn kinase activity in erythrocytes from ChAc patients were identified. How this contributes to the mechanism of neurodegeneration is still unknown. To gain insight into the pathophysiology, we established a ChAc patient-derived induced pluripotent stem cell model and an efficient differentiation protocol providing a large population of human striatal medium spiny neurons (MSNs), the main target of neurodegeneration in ChAc. Patient-derived MSNs displayed enhanced neurite outgrowth and ramification, whereas synaptic density was similar to controls. Electrophysiological analysis revealed a pathologically elevated synaptic activity in ChAc MSNs. Treatment with the F-actin stabilizer phallacidin or the Src kinase inhibitor PP2 resulted in the significant reduction of disinhibited synaptic currents to healthy control levels, suggesting a Src kinase- and actin-dependent mechanism. This was underlined by increased G/F-actin ratios and elevated Lyn kinase activity in patient-derived MSNs. These data indicate that F-actin stabilization and Src kinase inhibition represent potential therapeutic targets in ChAc that may restore neuronal function.SIGNIFICANCE STATEMENTChorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a humanin vitromodel using skin biopsies from ChAc patients to generate disease-specific induced pluripotent stem cells (iPSCs) and developed an efficient iPSC differentiation protocol providing striatal medium spiny neurons. Using patch-clamp electrophysiology, we detected a pathologically enhanced synaptic activity in ChAc neurons. Healthy control levels of synaptic activity could be restored by treatment of ChAc neurons with the F-actin stabilizer phallacidin and the Src kinase inhibitor PP2. Because Src kinases are involved in bridging the membrane to the actin cytoskeleton by membrane protein phosphorylation, our data suggest an actin-dependent mechanism of this dysfunctional phenotype and potential treatment targets in ChAc.

Background & Aims The addition of protease inhibitors, boceprevir (BOC) or telaprevir (TRV), to peg-interferon and ribavirin (PR) increases the incidence of anaemia in patients with chronic hepatitis C virus (HCV) infection. Although genetic variants in inosine triphosphatase (ITPA) gene have been linked to the haemolytic anaemia induced by PR, the mechanism sustaining severe anaemia during triple therapy is still unknown. This study aims to elucidate the molecular mechanisms underlying anaemia in chronic HCV patients with combined therapy. Methods We studied 59 patients with chronic HCV genotype-1: 29 treated with TRV/PR and 30 with BOC/PR. We evaluated biochemical and haematological parameters, red cell index at baseline, 4, 12, 16 and 24 weeks of treatment; in a subgroup, we performed functional studies: osmotic fragility, red cell membrane protein separation, mass spectrometry analysis, quantification of erythroid microparticles release. IL28B and ITPA polymorphisms were also evaluated. Results We found early acute normochromic normocytic haemolytic anaemia (4–8 weeks) followed by a late macrocytic hypo-regenerative anaemia with inappropriate low reticulocyte count (12–24 weeks). Studies on red cells revealed: (i) presence of spherocytes; (ii) increased osmotic fragility; (iii) abnormalities in red cell membrane protein composition; (iv) reduced membrane-cytoskeleton stability; (v) increased release of erythroid microparticles. ITPA polymorphisms impacted only the early phase of anaemia. Conclusions The bimodal pattern of anaemia in chronic HCV patients on triple therapy might be because of acquired spherocytic-like anaemia in the early phase, followed by hyporegenerative anaemia, most likely related to the combined effects of PR and TRV or BOC on erythropoiesis.