The Peptidoglycan Recognition Protein 1 confers immune evasive properties on pancreatic cancer stem cells.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending how this subset of cells evades the immune system is crucial for advancing novel therapies. DESIGN: We used the KPC mouse model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) and primary tumour cell lines to investigate putative CSC populations. Transcriptomic analyses were conducted to pinpoint new genes involved in immune evasion. Overexpressing and knockout cell lines were established with lentiviral vectors. Subsequent in vitro coculture assays, in vivo mouse and zebrafish tumorigenesis studies, and in silico database approaches were performed. RESULTS: Using the KPC mouse model, we functionally confirmed a population of cells marked by EpCAM, Sca-1 and CD133 as authentic CSCs and investigated their transcriptional profile. Immune evasion signatures/genes, notably the gene peptidoglycan recognition protein 1 (PGLYRP1), were significantly overexpressed in these CSCs. Modulating PGLYRP1 impacted CSC immune evasion, affecting their resistance to macrophage-mediated and T-cell-mediated killing and their tumourigenesis in immunocompetent mice. Mechanistically, tumour necrosis factor alpha (TNFα)-regulated PGLYRP1 expression interferes with the immune tumour microenvironment (TME) landscape, promoting myeloid cell-derived immunosuppression and activated T-cell death. Importantly, these findings were not only replicated in human models, but clinically, secreted PGLYRP1 levels were significantly elevated in patients with PDAC. CONCLUSIONS: This study establishes PGLYRP1 as a novel CSC-associated marker crucial for immune evasion, particularly against macrophage phagocytosis and T-cell killing, presenting it as a promising target for PDAC immunotherapy.

Platinum iodido drugs show potential anti-tumor activity, affecting cancer cell metabolism and inducing ROS and senescence in gastrointestinal cancer cells.

Cisplatin-based chemotherapy has associated clinical disadvantages, such as high toxicity and resistance. Thus, the development of new antitumor metallodrugs able to overcome different clinical barriers is a public healthcare priority. Here, we studied the mechanism of action of the isomers trans and cis-[PtI2(isopropylamine)2] (I5 and I6, respectively) against gastrointestinal cancer cells. We demonstrate that I5 and I6 modulate mitochondrial metabolism, decreasing OXPHOS activity and negatively affecting ATP-linked oxygen consumption rate. Consequently, I5 and I6 generated Reactive Oxygen Species (ROS), provoking oxidative damage and eventually the induction of senescence. Thus, herein we propose a loop with three interconnected processes modulated by these iodido agents: (i) mitochondrial dysfunction and metabolic disruptions; (ii) ROS generation and oxidative damage; and (iii) cellular senescence. Functionally, I5 reduces cancer cell clonogenicity and tumor growth in a pancreatic xenograft model without systemic toxicity, highlighting a potential anticancer complex that warrants additional pre-clinical studies.

Targeting cancer stem cell OXPHOS with tailored ruthenium complexes as a new anti-cancer strategy.

BACKGROUND: Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking. METHODS: The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action. RESULTS: We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration. CONCLUSIONS: Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Schistosomiasis screening in non-endemic countries from a cost perspective: Knowledge gaps and research priorities. The case of African long-term residents in a Metropolitan Area, Spain.

BACKGROUND: Imported schistosomiasis is an emerging issue in European countries as a result of growing global migration from schistosomiasis-endemic countries, mainly in sub-Saharan Africa. Undetected infection may lead to serious long-term complications with an associated high cost for public healthcare systems especially among long-term migrants. OBJECTIVE: To evaluate from a health economics perspective the introduction of schistosomiasis screening programs in non-endemic countries with high prevalence of long-term migrants. METHODOLOGY: We calculated the costs associated with three approaches-presumptive treatment, test-and-treat and watchful waiting-under different scenarios of prevalence, treatment efficacy and the cost of care resulting from long-term morbidity. Costs were estimated for our study area, in which there are reported to reside 74,000 individuals who have been exposed to the infection. Additionally, we methodically reviewed the potential factors that could affect the cost/benefit ratio of a schistosomiasis screening program and need therefore to be ascertained. RESULTS: Assuming a 24% prevalence of schistosomiasis in the exposed population and 100% treatment efficacy, the estimated associated cost per infected person of a watchful waiting strategy would be €2,424, that of a presumptive treatment strategy would be €970 and that of a test-and-treat strategy would be €360. The difference in averted costs between test-and-treat and watchful waiting strategies ranges from nearly €60 million in scenarios of high prevalence and treatment efficacy, to a neutral costs ratio when these parameters are halved. However, there are important gaps in our understanding of issues such as the efficacy of treatment in infected long-term residents, the natural history of schistosomiasis in long-term migrants and the feasibility of screening programs. CONCLUSION: Our results support the roll-out of a schistosomiasis screening program based on a test-and-treat strategy from a health economics perspective under the most likely projected scenarios, but important knowledge gaps should be addressed for a more accurate estimations among long-term migrants.

An Aptamer against MNK1 for Non-Small Cell Lung Cancer Treatment.

Lung cancer is the leading cause of cancer-related death worldwide. Its late diagnosis and consequently poor survival make necessary the search for new therapeutic targets. The mitogen-activated protein kinase (MAPK)-interacting kinase 1 (MNK1) is overexpressed in lung cancer and correlates with poor overall survival in non-small cell lung cancer (NSCLC) patients. The previously identified and optimized aptamer from our laboratory against MNK1, apMNKQ2, showed promising results as an antitumor drug in breast cancer in vitro and in vivo. Thus, the present study shows the antitumor potential of apMNKQ2 in another type of cancer where MNK1 plays a significant role, such as NSCLC. The effect of apMNKQ2 in lung cancer was studied with viability, toxicity, clonogenic, migration, invasion, and in vivo efficacy assays. Our results show that apMNKQ2 arrests the cell cycle and reduces viability, colony formation, migration, invasion, and epithelial-mesenchymal transition (EMT) processes in NSCLC cells. In addition, apMNKQ2 reduces tumor growth in an A549-cell line NSCLC xenograft model. In summary, targeting MNK1 with a specific aptamer may provide an innovative strategy for lung cancer treatment.

Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma.

OBJECTIVE: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models. DESIGN: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation. RESULTS: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment. CONCLUSION: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.

Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer.

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity in vitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.

Remdesivir in kidney transplant patients with SARS-CoV-2 pneumonia.

BACKGROUND: Remdesivir is the only antiviral treatment that has been shown to be useful against SARS-CoV-2 infection. It shorts hospitalization time compared to placebo. Its effects in Kidney transplant (KT) patients are limited to some published cases. METHODS: We performed a retrospective observational study that included all KT patients admitted between August 01, 2020 and December 31, 2020 with SARS-CoV-2 pneumonia who received remdesivir. The objective of this study was to describe the experience of a cohort of KT patients treated with remdesivir. DISCUSSION: A total of 37 KT patients developed SARS-CoV-2 infection, 7 of them received treatment with remdesivir. The rest of the patients did not receive the drug due to either CKD-EPI less than 30 mL/min or they did not present clinical criteria. In addition to remdesivir, all pacients received dexamethasone and anticoagulation therapy. 4 were men, the median age was 59 (53-71) years. Median time from transplantation was 43 (16-82) months. Chest X-rays of all patients showed pulmonary infiltrates and required low oxygen flow therapy upon admission, requiring high flow nasal therapy in 3 cases. Only 2 cases presented deterioration of the graft function, not requiring hemodialysis in any case, and all recovered renal function at hospital discharge. 2 patients rise up 1.5 times the liver function test. No patient died or required admission to the critical care unit. Median days of admission was 12 (9-27) days. CONCLUSIONS: Our study suggests that the use of remdesivir could be useful in KT patients with SARS-CoV-2 pneumonia without side effects. Additional studies are necessary with a larger number of patients to improve the knowledge of this drug in SARS-CoV-2 infection.

[Remdesivir in kidney transplant patients with SARS-CoV-2 pneumonia]. / Remdesivir en pacientes trasplantados renales con neumonía por SARS-CoV-2.

Background: Remdesivir is the only antiviral treatment that has been shown to be useful against SARS-CoV-2 infection. It shorts hospitalization time compared to placebo. Its effects in kidney transplant (KT) patients are limited to some published cases. Methods: We performed a retrospective observational study that included all KT patients admitted between August 1st, 2020 and December 31st, 2020 with SARS-CoV-2 pneumonia who received remdesivir.The objective of this study was to describe the experience of a cohort of KT patients treated with remdesivir. Discussion: A total of 37 KT patients developed SARS-CoV-2 infection, 7 of them received treatment with remdesivir. The rest of the patients did not receive the drug due to either CKD-EPI less than 30 mL/min or they did not present clinical criteria. In addition to remdesivir, all patients received dexamethasone and anticoagulation therapy. 4 were men, the median age was 59 (53-71) years. Median time from transplantation was 43 (16-82) months. Chest X-rays of all patients showed pulmonary infiltrates and required low-oxygen flow therapy upon admission, requiring high-flow nasal therapy in 3 cases. Only 2 cases presented deterioration of the graft function, not requiring hemodialysis in any case, and all recovered renal function at hospital discharge. 2 patients rise up 1.5 times the liver function test. No patient died or required admission to the critical care unit. Median days of admission was 12 (9-27) days. Conclusions: Our study suggests that the use of remdesivir could be useful in KT patients with SARS-CoV-2 pneumonia without side effects. Additional studies are necessary with a larger number of patients to improve the knowledge of this drug in SARS-CoV-2 infection.

Routine Biomarkers for the Severity of COVID-19 Pneumonia May Present Differently in Kidney Transplant Recipients.

BACKGROUND: The treatment of coronavirus disease 2019 (COVID-19) is based on the patient's clinical status and levels of inflammatory biomarkers. The comparative activity of these biomarkers in kidney transplant (KT) patients with COVID-19 pneumonia from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and non-SARS-CoV-2 etiologies is unknown. The aim of this study was to compare the clinical presentation and inflammatory parameters at admission of KT patients with COVID-19 pneumonia and those with non-COVID-19 pneumonia over the same period. METHODS: Biomarkers were measured and compared between KT patients with COVID-19 pneumonia (n = 57) and non-COVID-19 pneumonia (n = 20) from March 2020 to March 2021. RESULTS: Both groups showed comparable demographics. The KT patients with COVID-19 had fewer neutrophils (6824 ± 5000 vs 8969 ± 4206; P = .09) than the non-COVID group, although there was no significant difference in the lymphocyte count. Non-COVID-19 pneumonia was associated with higher d-dimer (median, 921 [interquartile range (IQR), 495-1680] vs median, 2215 [IQR, 879-3934]; P = 0.09) and interleukin-6 (median, 35 [IQR, 20-128] vs median, 222 [IQR, 38-500]; P = 0.006) levels. The ferritin level was higher in the COVID-19 group (median, 809 [IQR, 442-1,330] vs median, 377 [IQR, 276-885]; P = 0.008). In multivariable analysis, only d-dimer (hazard ratio [HR], 1; 95% confidence interval [CI],1-1.002; P = .02) and ferritin (HR, 1; 95% CI, 0.9-0.9; P = .02) increase the statistic signification. CONCLUSION: COVID-19 pneumonia in KT patients shows a different presentation of inflammatory biomarkers than other non-COVID pneumonias. It could be useful to identify KT patients with COVID-19. More detailed studies are necessary to understand the presentation of biomarkers in KT with COVID-19.

The Spanish Version of the Fear of Kidney Failure Questionnaire: Validity, Reliability, and Characterization of Living Donors With the Highest Fear of Kidney Failure.

BACKGROUND: Measures of fear of progression or recurrence of illnesses have been criticized for neglecting cross-cultural validity. Therefore, we assessed the psychometric properties of the Spanish version of the Fear of Kidney Failure Questionnaire (FKFQ), to determine whether postdonation fear of kidney failure (FKF) influenced the donors' psychosocial status, and define variables that characterized donors with high FKFQ scores. METHODS: We included 492 participants (211 donors) in a multicenter, 11-year, retrospective, cross-sectional study. Donors were classified with a Latent Class Analysis of the FKFQ-item scores and characterized with a multivariable logistic regression analysis. We calculated the risk ratio based on predicted marginal probabilities. RESULTS: The Spanish version of the FKFQ showed acceptable psychometric properties. FKF was uncommon among donors, but we detected a small subgroup (n = 21, 9.9%) with high FKF (mean FKFQ score = 14.5, 3.1 SD). Compared with other donors, these donors reported higher anxiety and depression (38% and 29% of potential anxiety and depressive disorders), worse quality of life, and less satisfaction with the donation. Donors with high FKFQ scores were characterized by higher neuroticism combined with postdonation physical symptoms that interfered with daily activities. CONCLUSIONS: The FKFQ was cross-culturally valid, and thus, it may be used to assess the FKF in Spanish-speaking donors. New interventions that promote positive affectivity and evidence-based treatments for worry could be adapted for treating FKF.

Urinary vitronectin identifies patients with high levels of fibrosis in kidney grafts.

BACKGROUND: In kidney transplantation, fibrosis represents the final and irreversible consequence of the pathogenic mechanisms that lead to graft failure, and in the late stages it irremediably precedes the loss of renal function. The invasiveness of kidney biopsy prevents this condition from being frequently monitored, while clinical data are rather unspecific. The objective of this study was to find noninvasive biomarkers of kidney rejection. METHODS: We carried out proteomic analysis of the urinary Extracellular Vesicles (uEVs) from a cohort of kidney transplant recipients (n = 23) classified according to their biopsy-based diagnosis and clinical parameters as interstitial fibrosis and tubular atrophy (IFTA), acute cellular rejection (ACR), calcineurin inhibitors toxicity (CNIT) and normal kidney function (NKF). RESULTS: Shotgun mass spectrometry of uEV-proteins identified differential expression of several proteins among these different groups. Up to 23 of these proteins were re-evaluated using targeted proteomics in a new independent cohort of patients (n = 41) classified in the same diagnostic groups. Among other results, we found a differential expression of vitronectin (VTN) in patients displaying chronic interstitial and tubular lesions (ci and ct mean > 2 according to Banff criteria). These results were further confirmed by a pilot study using enzyme-linked immunosorbent assay (ELISA). CONCLUSION: Urinary vitronectin levels are a potential stand-alone biomarker to monitor fibrotic changes in kidney transplant recipients in a non-invasive fashion.

Avoidant/restrictive food intake disorder: Psychopathological similarities and differences in comparison to anorexia nervosa and the general population.

INTRODUCTION: Avoidant/restrictive food intake disorder (ARFID) categorises patients with selective and/or restrictive eating patterns in the absence of distorted cognition concerning weight, food, and body image. OBJECTIVE: To examine the sociodemographic and clinical profile of patients with ARFID in comparison to those with anorexia nervosa (AN) and to a non-clinical group (NCG). METHOD: A descriptive, observational, comparative study made up of three groups (ARFID, AN and NCG). Ninety-nine children and adolescents were analyzed by means of a semi-structured diagnostic interview and questionnaires on depression, anxiety, clinical fears and general psychopathology. RESULTS: The ARFID group was significantly younger (10.8 vs. 14.1 years of age), with a greater proportion of males (60.6% vs. 6.1%), an earlier onset of illness (6.2 vs. 13.4 years of age), and a longer period of evolution of the illness (61.2 vs. 8.4 months) compared to the AN group. Clinically, patients with ARFID showed greater medical (42.4% vs. 12.1%) and psychiatric (81.8% vs. 33.3%) comorbidity-assessed with a semi-structured diagnostic interview-greater clinical fear (p < 0.005), more attention problems (p < 0.005) and fewer symptoms of anxiety and depression (p < 0.005)-measured with self-report questionnaires. CONCLUSIONS: ARFID is a serious disorder with a significant impact on the physical and mental health of the pediatric population. Likewise, some of these physical and mental conditions may be a risk factor in developing ARFID. Attention problems and clinical fears in ARFID, and the greater presence of internalised symptoms in AN, were the main differences found in the psychopathological profiles.

Correction: Alcalá, S., et al. Targeting SRC Kinase Signaling in Pancreatic Cancer Stem Cells. Int. J. Mol. Sci. 2020, 21, 7437.

The authors recently reported on the potential of targeting SRC kinase signaling in pancreatic cancer stem cells [...].

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells.

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7-9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.

Proteomic Characterization of Urinary Extracellular Vesicles from Kidney-Transplanted Patients Treated with Calcineurin Inhibitors.

Use of immunosuppressive drugs is still unavoidable in kidney-transplanted patients. Since their discovery, calcineurin inhibitors (CNI) have been considered the first-line immunosuppressive agents, in spite of their known nephrotoxicity. Chronic CNI toxicity (CNIT) may lead to kidney fibrosis, a threatening scenario for graft survival. However, there is still controversy regarding CNIT diagnosis, monitoring and therapeutic management, and their specific effects at the molecular level are not fully known. Aiming to better characterize CNIT patients, in the present study, we collected urine from kidney-transplanted patients treated with CNI who (i) had a normal kidney function, (ii) suffered CNIT, or (iii) presented interstitial fibrosis and tubular atrophy (IFTA). Urinary extracellular vesicles (uEV) were enriched and the proteome was analyzed to get insight into changes happening during CNI. Members of the uroplakin and plakin families were significantly upregulated in the CNIT group, suggesting an important role in CNIT processes. Although biomarkers cannot be asserted from this single pilot study, our results evidence the potential of uEV as a source of non-invasive protein biomarkers for a better detection and monitoring of this renal alteration in kidney-transplanted patients.

Targeting SRC Kinase Signaling in Pancreatic Cancer Stem Cells.

The proto-oncogene nonreceptor tyrosine-protein kinase SRC is a member of the SRC family of tyrosine kinases (SFKs), and its activation and overexpression have been shown to play a protumorigenic role in multiple solid cancers, including pancreatic ductal adenocarcinoma (PDAC). PDAC is currently the seventh-leading cause of cancer-related death worldwide, and, by 2030, it is predicted to become the second-leading cause of cancer-related death in the United States. PDAC is characterized by its high lethality (5-year survival of rate of <10%), invasiveness, and chemoresistance, all of which have been shown to be due to the presence of pancreatic cancer stem cells (PaCSCs) within the tumor. Due to the demonstrated overexpression of SRC in PDAC, we set out to determine if SRC kinases are important for PaCSC biology using pharmacological inhibitors of SRC kinases (dasatinib or PP2). Treatment of primary PDAC cultures established from patient-derived xenografts with dasatinib or PP2 reduced the clonogenic, self-renewal, and tumor-initiating capacity of PaCSCs, which we attribute to the downregulation of key signaling factors such as p-FAK, p-ERK1-2, and p-AKT. Therefore, this study not only validates that SRC kinases are relevant and biologically important for PaCSCs but also suggests that inhibitors of SRC kinases may represent a possible future treatment option for PDAC patients, although further studies are still needed.

ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity.

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness.

Molecular profile of urine extracellular vesicles from normo-functional kidneys reveal minimal differences between living and deceased donors.

BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney diseases leading to irreversible kidney failure. Considering the origin of the graft, several studies have reported differences between living (LD) and deceased donors (DD) in graft and patient survival. These differences seem to be related to multiple factors including, donor age and time of cold ischemia among others. Many of transplanted organs come from old-aged DDs, in which pre-transplant biopsy is recommended. However, kidney biopsy has several limitations, and there is a need to develop alternatives to assess the status of a kidney before transplantation. As the analysis of urinary extracellular vesicles (uEVs) rendered promising results as non-invasive biomarkers of kidney-related pathologies, this pilot study aimed to investigate whether profiling uEVs of LDs and DDs may be of help to assess the quality of the kidney before nephrectomy. METHODS: uEVs from 5 living donors and 7 deceased donors were isolated by size-exclusion chromatography, and their protein and miRNA content were analysed by liquid chromatography followed by mass spectrometry and next generation sequencing, respectively. Then, hierarchical clustering and venn diagrams were done with Perseus software and InteractiVenn tool. Specific EVs data bases were also used for Gene Ontology analysis. RESULTS: Next generation sequencing revealed that uEVs from DDs contained less miRNAs than LDs, but most of the DD-expressed miRNAs were shared with LDs (96%). Only miR-326 (targeting the apoptotic-related Bcl2) was found significantly over-represented in LD. Focusing on the protein content, we detected a low intra-group correlation in both types of donors. Despite these differences, hierarchical clustering of either miRNA or protein data could not identify a differential profile between LDs and DDs. Of note, 90% of transplanted patients had a functional graft after a year from KTx. CONCLUSIONS: In this pilot study we found that, in normo-functional grafts, minor differences in uEVs profile could not discriminate between LDs and DDs.