Distinct and overlapping functions of glutathione peroxidases 1 and 2 in limiting NF-κB-driven inflammation through redox-active mechanisms

Glutathione peroxidase 2 (GPx2) is one of the five selenoprotein GPxs having a selenocysteine in the active center. GPx2 is strongly expressed in the gastrointestinal epithelium, as is another isoform, GPx1, though with a different localization pattern. Both GPxs are redox-active enzymes that are important for the reduction of hydroperoxides. Studies on GPx2-deficient mice and human HT-29 cells with a stable knockdown (kd) of GPx2 revealed higher basal and IL-1β-induced expression of NF-κB target genes in vivo and in vitro. The activation of the IKK-IκBα-NF-κB pathway was increased in cultured GPx2 kd cells. Basal signaling was only restored by re-expressing active GPx2 in GPx2 kd cells but not by redox-inactive GPx2. As it is still not clear if the two isoforms GPx1 and GPx2 have different functions, kd cell lines for either GPx1 or GPx2 were studied in parallel. The inhibitory effect of GPx2 on NF-κB signaling and its target gene expression was stronger than that of GPx1, whereas cyclooxygenase (COX)- and lipoxygenase (LOX)-derived lipid mediator levels increased more strongly in GPx1 kd than in GPx2 kd cells. Under unstimulated conditions, the levels of the COX-derived prostaglandins PGE2 and PGD2 were enhanced in GPx2 as well as in GPx1 kd compared to control cells. Specifically, in GPx1 kd cells IL-1β stimulation led to a dramatic shift of the PGE2/PGD2 ratio towards pro-inflammatory PGE2. Taken together, GPx2 and GPx1 have overlapping functions in controlling inflammatory lipid mediator synthesis and, most probably, exert their anti-inflammatory effects by preventing excessive PGE2 production. In view of the high activity of COX and LOX pathways during inflammatory bowel disease our data therefore provide new insights into the mechanisms of the protective function of GPx1 and GPx2 during colitis as well as inflammation-driven carcinogenesis

BRAFV600E in colorectal cancer reduces sensitivity to oxidative stress and promotes site-specific metastasis by stimulating glutathione synthesis

The presence of BRAFV600E in colorectal cancer (CRC) is associated with a higher chance of distant metastasis. Oxidative stress in disseminated tumor cells limits metastatic capacity. To study the relationship between BRAFV600E, sensitivity to oxidative stress, and metastatic capacity in CRC, we use patient-derived organoids (PDOs) and tissue samples. BRAFV600E tumors and PDOs express high levels of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione synthesis. Deletion of GCL in BRAFV600E PDOs strongly reduces their capacity to form distant liver and lung metastases but does not affect peritoneal metastasis outgrowth. Vice versa, the glutathione precursor N-acetyl-cysteine promotes organ-site-specific metastasis in the liver and the lungs but not in the peritoneum. BRAFV600E confers resistance to pharmacologically induced oxidative stress in vitro, which is partially overcome by treatment with the BRAF-inhibitor vemurafenib. We conclude that GCL-driven glutathione synthesis protects BRAFV600E-expressing tumors from oxidative stress during distant metastasis to the liver and the lungs

Modeling resistance of colorectal peritoneal metastases to immune checkpoint blockade in humanized mice

Background The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. Methods We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-Associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. Results PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γsignature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. Conclusions By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB

Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer

BACKGROUND: Poor prognosis in colon cancer is associated with a high content of cancer-associated fibroblasts (CAFs) and an immunosuppressive tumor microenvironment. The relationship between these two features is incompletely understood. Here, we aimed to generate a model system for studying the interaction between cancer cells and CAFs and their effect on immune-related cytokines and T cell proliferation. METHODS: CAFs were isolated from colon cancer liver metastases and were immortalized to prolong lifespan and improve robustness and reproducibility. Established medium and matrix compositions that support the growth of patient-derived organoids were adapted to also support CAF growth. Changes in growth pattern and cellular re-organization were assessed by confocal microscopy, live cell imaging, and immunofluorescence. Single cell RNA sequencing was used to study CAF/organoid co-culture-induced phenotypic changes in both cell types. Conditioned media were used to quantify the production of immunosuppressive factors and to assess their effect on T cell proliferation. RESULTS: We developed a co-culture system in which colon cancer organoids and CAFs spontaneously organize into superstructures with a high capacity to contract and stiffen the extracellular matrix (ECM). CAF-produced collagen IV provided a basement membrane supporting cancer cell organization into glandular structures, reminiscent of human cancer histology. Single cell RNA sequencing analysis showed that CAFs induced a partial epithelial-to-mesenchymal-transition in a subpopulation of cancer cells, similar to what is observed in the mesenchymal-like consensus molecular subtype 4 (CMS4) colon cancer. CAFs in co-culture were characterized by high expression of ECM components, ECM-remodeling enzymes, glycolysis, hypoxia, and genes involved in immunosuppression. An expression signature derived from CAFs in co-culture identified a subpopulation of glycolytic myofibroblasts specifically residing in CMS1 and CMS4 colon cancer. Medium conditioned by co-cultures contained high levels of the immunosuppressive factors TGFβ1, VEGFA and lactate, and potently inhibited T cell proliferation. CONCLUSION: Co-cultures of organoids and immortalized CAFs recapitulate the histological, biophysical, and immunosuppressive features of aggressive mesenchymal-like human CRC. The model can be used to study the mechanisms of immunosuppression and to test therapeutic strategies targeting the cross-talk between CAFs and cancer cells. It can be further modified to represent distinct colon cancer subtypes and (organ-specific) microenvironments

Lymphatic Invasion of Plakoglobin-Dependent Tumor Cell Clusters Drives Formation of Polyclonal Lung Metastases in Colon Cancer

Background & Aims: Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. Methods: Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, in vitro, and in vivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. Results: Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. Conclusions: Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site

ETUDE DE LA DIFFERENCIATION MUCOCILIAIRE DES CELLULES EPITHELIALES RESPIRATOIRES DES MAMMIFERES (MODULATION PAR L'INTERLEUKINE 13 ET EXPRESSION DES CENTRINES)

L'EPITHELIUM DES VOIES RESPIRATOIRES SUPERIEURES EST LA PREMIERE CIBLE DES FACTEURS EXOGENES AERIENS (MOLECULES, PARTICULES, AGENTS INFECTIEUX). DES LESIONS QUI PEUVENT AFFECTER L'INTEGRITE DE CETTE BARRIERE NECESSITENT LE RENOUVELLEMENT RAPIDE DES CELLULES DIFFERENCIEES, CILIEES ET SECRETRICES. PAR AILLEURS, CES LESIONS ENGENDRENT UN PROCESSUS INFLAMMATOIRE DU A L'ACCUMULATION ET L'ACTIVATION AU NIVEAU DE LA MUQUEUSE RESPIRATOIRE DE CELLULES INFLAMMATOIRES. CES CELLULES VONT LIBERER PLUSIEURS MEDIATEURS D'INFLAMMATION QUI SONT EN PARTIE RESPONSABLES DE L'ALTERATION DU TISSU EPITHELIAL. LA COMPREHENSION DE LA REGULATION DE LA DIFFERENCIATION MUCOCILIAIRE EN AMONT (INFLUENCE DE FACTEURS EXTERIEURS TELS QUE LES CYTOKINES) ET EN AVAL (MARQUEURS DE DIFFERENCIATION, REGULATEURS INTRACELLULAIRES), EST DE FAIT UN ENJEU MAJEUR POUR LA CONNAISSANCE DU TISSU NORMAL ET PATHOLOGIQUE. NOUS AVONS DEVELOPPE PLUSIEURS MODELES DE CULTURE PRIMAIRE DE CELLULES EPITHELIALES DES VOIES RESPIRATOIRES SUPERIEURES CHEZ LES MAMMIFERES QUI PERMETTENT D'INDUIRE UNE DIFFERENCIATION MUCOCILIAIRE IN VITRO, ET NOUS AVONS QUANTIFIE CE PROCESSUS. DANS UN MODELE HUMAIN, NOUS AVONS CARACTERISE AU COURS DE LA CILIOGENESE UNE FAMILLE DE PROTEINES CENTRIOLAIRES, LES CENTRINES, APPARTENANT A LA SUPER-FAMILLE DES PROTEINES LIANT LE CALCIUM. L'ETUDE DES TROIS ISOFORMES DE CENTRINES MONTRE UNE AUGMENTATION IMPORTANTE DE LEUR EXPRESSION AU COURS DE LA DIFFERENCIATION, ET UNE LOCALISATION PRECOCE ET RESTREINTE DANS LES STRUCTURES PRECURSEURS DES CENTRIOLES, SUGGERANT UN ROLE DE CES PROTEINES DANS L'ASSEMBLAGE DES CENTRIOLES. DES DONNEES IMMUNOCYTOCHIMIQUES REVELENT UNE DISTRIBUTION DIFFERENTE DES TROIS ISOFORMES AU NIVEAU DES CORPS BASAUX ET DES AXONEMES CILIAIRES DES CELLULES EPITHELIALES CILIEES. LEUR DIFFERENCE DE LOCALISATION EST CORRELEE AVEC UN ROLE SPECIFIQUE DE LA CENTRINE 1/2 DANS L'ACTIVITE DE BATTEMENT CILIAIRE SUGGERANT DES FONCTIONS CELLULAIRES DIFFERENTES DES CENTRINES HUMAINES DANS LES CELLULES EPITHELIALES RESPIRATOIRES. DANS LA DEUXIEME PARTIE DE CE TRAVAIL, NOUS AVONS ANALYSE L'EFFET DE L'INTERLEUKINE 13 (IL-13) AU COURS DE LA DIFFERENCIATION MUCOCILIAIRE. L'IL-13 EST UNE CYTOKINE PRODUITE PRINCIPALEMENT PAR LES LYMPHOCYTES T DE TYPE TH2 QUI JOUE UN ROLE CLE DANS LES MANIFESTATIONS ALLERGIQUES RESPIRATOIRES TELLES QUE L'ASTHME. NOUS AVONS MONTRE QUE L'IL-13 DIMINUAIT LA PROPORTION DES CELLULES CILIEES, ET AUGMENTAIT DE FACON IMPORTANTE LA PROPORTION DES CELLULES SECRETRICES AU COURS DE LA DIFFERENCIATION IN VITRO. L'IL-13 PERTURBE EGALEMENT L'ORGANISATION DES CELLULES EPITHELIALES ET ALTERE LA DISTRIBUTION DE MARQUEURS DE POLARISATION TELS QUE L'EZRINE. UNE ANALYSE DIFFERENTIELLE D'ARN MESSAGERS MONTRE QUE L'IL-13 MODIFIE L'EXPRESSION DE NOMBREUX GENES. EN PARTICULIER, ELLE INHIBE L'EXPRESSION DE LA P73, PROTEINE PRESENTANT UNE FORTE HOMOLOGIE DE SEQUENCE AVEC LE PRODUIT DU GENE SUPPRESSEUR DE TUMEUR P53. SON EXPRESSION DIFFERENTIELLE AU COURS DE LA PROLIFERATION ET LA DIFFERENCIATION DES CELLULES EPITHELIALES RESPIRATOIRES SUGGERE UN ROLE POTENTIEL DE LA P73 DANS LA DIFFERENCIATION MUCOCILIAIRE.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Loss of Neuropilin-2 in Murine Mesenchymal-like Colon Cancer Organoids Causes Mesenchymal-to-Epithelial Transition and an Acquired Dependency on Insulin-Receptor Signaling and Autophagy

Neuropilin-2 (Nrp2), an important regulator of lymphangiogenesis and lymphatic metastasis, has been associated with progression in colorectal cancer (CRC). However, the tumor cell-intrinsic role of Nrp2 in cancer progression is incompletely understood. To address this question, we employed CRISPR-Cas9 technology to generate Nrp2-knockout organoids derived from murine CRC tumors with a mesenchymal phenotype. Transcriptome profiling and tumor tissue analysis showed that Nrp2 loss resulted in mesenchymal-to-epithelial transition (MET), which was accompanied with restored polarity and tight junction stabilization. Signaling pathway analysis revealed that Nrp2-knockout organoids acquire de novo dependency on insulin receptor (IR) signaling and autophagy as alternative survival mechanisms. Combined inhibition of IR signaling and autophagy prevented the stabilization of cell-cell junctions, reduced metabolic activity, and caused profound cell death in Nrp2-knockout organoids. Collectively, the data demonstrate a key role for Nrp2 in maintaining the aggressive phenotype and survival of tumor-derived CRC organoids. The identified connection between Nrp2, insulin receptor signaling and autophagy may guide the development of novel combination-treatment strategies for aggressive CRC