Cellular collusion: cracking the code of immunosuppression and chemo resistance in PDAC.

Despite the efforts, pancreatic ductal adenocarcinoma (PDAC) is still highly lethal. Therapeutic challenges reside in late diagnosis and establishment of peculiar tumor microenvironment (TME) supporting tumor outgrowth. This stromal landscape is highly heterogeneous between patients and even in the same patient. The organization of functional sub-TME with different cellular compositions provides evolutive advantages and sustains therapeutic resistance. Tumor progressively establishes a TME that can suit its own needs, including proliferation, stemness and invasion. Cancer-associated fibroblasts and immune cells, the main non-neoplastic cellular TME components, follow soluble factors-mediated neoplastic instructions and synergize to promote chemoresistance and immune surveillance destruction. Unveiling heterotypic stromal-neoplastic interactions is thus pivotal to breaking this synergism and promoting the reprogramming of the TME toward an anti-tumor milieu, improving thus the efficacy of conventional and immune-based therapies. We underscore recent advances in the characterization of immune and fibroblast stromal components supporting or dampening pancreatic cancer progression, as well as novel multi-omic technologies improving the current knowledge of PDAC biology. Finally, we put into context how the clinic will translate the acquired knowledge to design new-generation clinical trials with the final aim of improving the outcome of PDAC patients.

Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC.

OBJECTIVE: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DESIGN: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients' tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. RESULTS: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. CONCLUSIONS: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.

Performance in Behavioral Testing in an Animal Model of Post-Surgical Hypoparathyroidism.

BACKGROUND: Hypoparathyroidism (HypoPT) is characterized by hypocalcemia and undetectable/inappropriately low PTH. Post-surgical HypoPT (PS-HypoPT) is the most common cause. Patients with PS-HypoPT present neuropsychological symptoms, probably due to the PTH deprivation in the central nervous system (CNS). However, these mechanisms are still not elucidated. The aim of this study was to evaluate the effects of PTH deprivation on CNS in an animal model of PS-HypoPT via a cognitive/behavioral assessment approach. METHODS: A surgical rat model of PS-HypoPT was obtained and treated with calcium to maintain normocalcemia. Twenty PS-HypoPT rats and twenty sham-operated controls (Crl) underwent behavioral testing in a Morris Water Maze (MWM), Open Field (OF), and Elevated Plus Maze (EPM). RESULTS: In the MWM, PTx rats showed a higher Escape Latency Time compared to Crl rats (p < 0.05); we observed a statistically significant improvement in the performance (day 1 to 8 p < 0.001), which was less pronounced in PTx group. In the OF test, the time and distance spent in the zone of interest were significantly lower in the PTx group compared with the Crl (p < 0.01 and p < 0.01). In the EPM experiment, the time spent in the close arm was significantly higher in the PTx group compared with the Crl (p < 0.01). CONCLUSIONS: This animal model of PS-HypoPT shows an impairment in spatial memory, which improved after training, and a marked anxiety-like behavior, resembling the condition of patients with PS-HypoPT. Further studies are needed to elucidate mechanisms.

Loss of FGFR4 promotes the malignant phenotype of PDAC.

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.

The role of kidney dysfunction in COVID-19 and the influence of age.

COVID-19 is strongly influenced by age and comorbidities. Acute kidney injury (AKI) is a frequent finding in COVID-19 patients and seems to be associated to mortality and severity. On the other hand, the role of kidney dysfunction in COVID-19 is still debated. We performed a retrospective study in a cohort of 174 hospitalized COVID-19 patients in Italy from March 3rd to May 21st 2020, to investigate the role of kidney dysfunction on COVID-19 severity and mortality. Moreover, we examined in depth the relationship between kidney function, age, and progression of COVID-19, also using different equations to estimate the glomerular filtration rate (GFR). We performed logistic regressions, while a predictive analysis was made through a machine learning approach. AKI and death occurred respectively in 10.2% and 19.5%, in our population. The major risk factors for mortality in our cohort were age [adjusted HR, 6.2; 95% confidence interval (CI) 1.8-21.4] and AKI [3.36 (1.44-7.87)], while, in these relationships, GFR at baseline mitigated the role of age. The occurrence of AKI was influenced by baseline kidney function, D-dimer, procalcitonin and hypertension. Our predictive analysis for AKI and mortality reached an accuracy of ≥ 94% and ≥ 91%, respectively. Our study scales down the role of kidney function impairment on hospital admission , especially in elderly patients. BIS-1 formula demonstrated a worse performance to predict the outcomes in COVID-19 patients when compared with MDRD and CKD-EPI.

Generation of Pancreatic Organoid-Derived Isografts.

This protocol is a procedure for generating orthotopic isografts using mouse pancreatic cancer organoids. These isografts can be used to track the evolution of pancreatic ductal adenocarcinoma (PDA) from a preinvasive lesion to a metastatic disease and therefore represent a suitable model for identification of determinants of PDA progression. For complete details on the use and execution of this protocol, please refer to Boj et al. (2015) and Filippini et al. (2019).

Fyn specifically Regulates the activity of red cell glucose-6-phosphate-dehydrogenase.

Fyn is a tyrosine kinase belonging to the Src family (Src-Family-Kinase, SFK), ubiquitously expressed. Previously, we report that Fyn is important in stress erythropoiesis. Here, we show that in red cells Fyn specifically stimulates G6PD activity, resulting in a 3-fold increase enzyme catalytic activity (kcat) by phosphorylating tyrosine (Tyr)-401. We found Tyr-401 on G6PD as functional target of Fyn in normal human red blood cells (RBC), being undetectable in G6PD deficient RBCs (G6PD-Mediterranean and G6PD-Genova). Indeed, Tyr-401 is located to a region of the G6PD molecule critical for the formation of the enzymatically active dimer. Amino acid replacements in this region are mostly associated with a chronic hemolysis phenotype. Using mutagenesis approach, we demonstrated that the phosphorylation status of Tyr401 modulates the interaction of G6PD with G6P and stabilizes G6PD in a catalytically more efficient conformation. RBCs from Fyn-/-mice are defective in G6PD activity, resulting in increased susceptibility to primaquine-induced intravascular hemolysis. This negatively affected the recycling of reduced Prx2 in response to oxidative stress, indicating that defective G6PD phosphorylation impairs defense against oxidation. In human RBCs, we confirm the involvement of the thioredoxin/Prx2 system in the increase vulnerability of G6PD deficient RBCs to oxidation. In conclusion, our data suggest that Fyn is an oxidative radical sensor, and that Fyn-mediated Tyr-401 phosphorylation, by increasing G6PD activity, plays an important role in the physiology of RBCs. Failure of G6PD activation by this mechanism may be a major limiting factor in the ability of G6PD deficient RBCs to withstand oxidative stress.

Organoid-Transplant Model Systems to Study the Effects of Obesity on the Pancreatic Carcinogenesis in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality among adults in developed countries. The discovery of the most common genetic alterations as well as the development of organoid models of pancreatic cancer have provided insight into the fundamental pathways driving tumor progression from a normal cell to non-invasive precursor lesion and finally to widely metastatic disease, offering new opportunities for identifying the key driver of cancer evolution. Obesity is one of the most serious public health challenges of the 21st century. Several epidemiological studies have shown the positive association between obesity and cancer-related morbidity/mortality, as well as poorer prognosis and treatment outcome. Despite strong evidence indicates a link between obesity and cancer incidence, the molecular basis of the initiating events remains largely elusive. This is mainly due to the lack of an accurate and reliable model of pancreatic carcinogenesis that mimics human obesity-associated PDAC, making data interpretation difficult and often confusing. Here we propose a feasible and manageable organoid-based preclinical tool to study the effects of obesity on pancreatic carcinogenesis. Therefore, we tracked the effects of obesity on the natural evolution of PDAC in a genetically defined transplantable model of the syngeneic murine pancreatic preneoplastic lesion (mP) and tumor (mT) derived-organoids that recapitulates the progression of human disease from early preinvasive lesions to metastatic disease. Our results suggest that organoid-derived transplant in obese mice represents a suitable system to study early steps of pancreatic carcinogenesis and supports the hypothesis that inflammation induced by obesity stimulates tumor progression and metastatization during pancreatic carcinogenesis.

Immunoevolution of mouse pancreatic organoid isografts from preinvasive to metastatic disease.

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.

Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization.

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 STATEMENT: Chorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a human in vitro model 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.

A new molecular link between defective autophagy and erythroid abnormalities in chorea-acanthocytosis.

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.

Protease inhibitors-based therapy induces acquired spherocytic-like anaemia and ineffective erythropoiesis in chronic hepatitis C virus patients.

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.

The novel role of peroxiredoxin-2 in red cell membrane protein homeostasis and senescence.

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.

UV-Induced Radical Photo-Polymerization: A Smart Tool for Preparing Polymer Electrolyte Membranes for Energy Storage Devices.

In the present work, the preparation and characterization of quasi-solid polymer electrolyte membranes based on methacrylic monomers and oligomers, with the addition of organic plasticizers and lithium salt, are described. Noticeable improvements in the mechanical properties by reinforcement with natural cellulose hand-sheets or nanoscale microfibrillated cellulose fibers are also demonstrated. The ionic conductivity of the various prepared membranes is very high, with average values approaching 10-3 S cm-1 at ambient temperature. The electrochemical stability window is wide (anodic breakdown voltages > 4.5 V vs. Li in all the cases) along with good cyclability in lithium cells at ambient temperature. The galvanostatic cycling tests are conducted by constructing laboratory-scale lithium cells using LiFePO4 as cathode and lithium metal as anode with the selected polymer electrolyte membrane as the electrolyte separator. The results obtained demonstrate that UV induced radical photo-polymerization is a well suited method for an easy and rapid preparation of easy tunable quasi-solid polymer electrolyte membranes for energy storage devices.

A psychosocial cancer phone center staffed by professional psychologists as an integral part of the standard process of care: Its utility during the course of illness.

OBJECTIVE: Major cancer centers cannot ignore psychosocial patient needs that have a significant impact on the process of adjustment to cancer and on compliance to treatments. We introduced a new service, the Gigi Ghirotti Psychosocial Cancer Phone Center (GGPCPC), staffed by professional psychologists, for use by our patients and their relatives. This article investigates its feasibility as a support delivery vehicle for patients in their follow-up phase, and also investigates patients' sense of abandonment related to their care setting. METHOD: A close collaboration was set up between GGPCPC psychologists and European Institute of Oncology (IEO) psychologists. Education and awareness sessions regarding the importance of such a source of psychological support were conducted by IEO psychologists with nurses, secretaries, and receptionists. IEO psychologists input monthly data, recorded on specific paper-tabs by GGPCPC psychologists for each call received by the phone center between March 2007 and March 2009, into a SPSS database. RESULTS: Four hundred and thirty individuals contacted the center mainly to receive psychological support during their treatment phase, when they visited IEO for treatment sessions. Multiple indicators suggest that this telephone support program was feasible and provided support to a broad range of cancer patients. Patients seemed to prefer it to face-to-face psychological support during their treatment-phase. SIGNIFICANCE OF RESULTS: The GGPCPC was demonstrated to be an efficacious support and information delivery vehicle for patients and relatives during the illness course.