The Nlrp3 Inflammasome Suppresses Colorectal Cancer Metastatic Growth in the Liver by Promoting Natural Killer Cell Tumoricidal Activity.

The crosstalk between inflammation and tumorigenesis is now clearly established. However, how inflammation is elicited in the metastatic environment and the corresponding contribution of innate immunity pathways in suppressing tumor growth at secondary sites are poorly understood. Here, we show that mice deficient in Nlrp3 inflammasome components had exacerbated liver colorectal cancer metastatic growth, which was mediated by impaired interleukin-18 (IL-18) signaling. Control of tumor growth was independent of differential cancer cell colonization or proliferation, intestinal microbiota effects, or tumoricidal activity by the adaptive immune system. Instead, the inflammasome-IL-18 pathway impacted maturation of hepatic NK cells, surface expression of the death ligand FasL, and capacity to kill FasL-sensitive tumors. Our results define a regulatory signaling circuit within the innate immune system linking inflammasome activation to effective NK-cell-mediated tumor attack required to suppress colorectal cancer growth in the liver.

Diabetes Impaired Ischemia-Induced PDGF (Platelet-Derived Growth Factor) Signaling Actions and Vessel Formation Through the Activation of Scr Homology 2-Containing Phosphatase-1.

Objective: Critical limb ischemia is a major complication of diabetes characterized by insufficient collateral vessel development and proper growth factor signaling unresponsiveness. Although mainly deactivated by hypoxia, phosphatases are important players in the deregulation of proangiogenetic pathways. Previously, SHP-1 (Scr homology 2-containing phosphatase-1) was found to be associated with the downregulation of growth factor actions in the diabetic muscle. Thus, we aimed to gain further understanding of the impact of SHP-1 on smooth muscle cell (SMC) function under hypoxic and diabetic conditions. Approach and Results: Despite being inactivated under hypoxic conditions, high glucose level exposure sustained SHP-1 phosphatase activity in SMC and increased its interaction with PDGFR (platelet-derived growth factor receptor)-ß, thus reducing PDGF proangiogenic actions. Overexpression of an inactive form of SHP-1 fully restored PDGF-induced proliferation, migration, and signaling pathways in SMC exposed to high glucose and hypoxia. Nondiabetic and diabetic mice with deletion of SHP-1 specifically in SMC were generated. Ligation of the femoral artery was performed, and blood flow was measured for 4 weeks. Blood flow reperfusion, vascular density and maturation, and limb survival were all improved while vascular apoptosis was attenuated in diabetic SMC-specific SHP-1 null mice as compared to diabetic mice. Conclusions: Diabetes and high glucose level exposure maintained SHP-1 activity preventing hypoxia-induced PDGF actions in SMC. Specific deletion of SHP-1 in SMC partially restored blood flow reperfusion in the diabetic ischemic limb. Therefore, local modulation of SHP-1 activity in SMC could represent a potential therapeutic avenue to improve the proangiogenic properties of SMC under ischemia and diabetes.

Identification of the bacteria and their metabolic activities associated with the microbial spoilage of custard cream desserts.

The famous French dessert "ile flottante" consists of a sweet egg white foam floating on a vanilla custard cream, which contains highly nutritive raw materials, including milk, sugar and egg. Spoilage issues are therefore a key concern for the manufacturers. This study explored the bacterial diversity of 64 spoiled custard cream desserts manufactured by 2 French companies. B. cereus group bacteria, coagulase negative Staphylococcus, Enterococcus and Leuconostoc spp. were isolated from spoiled products. Thirty-one bacterial isolates representative of the main spoilage species were tested for their spoilage abilities. Significant growth and pH decrease were observed regardless of species. While off-odours were detected with B. cereus group and staphylococci, yoghurt odours were detected with Enterococcus spp. and Leuconostoc spp. B. cereus group bacteria produced various esters and several compounds derived from amino acid and sugar metabolism. Most Staphylococci produced phenolic compounds. Enterococcus spp. and Leuconostoc spp. isolates produced high levels of compounds derived from sugar metabolism. Each type of spoilage bacteria was associated with a specific volatile profile and lactic acid was identified as a potential marker of spoilage of custard cream-based desserts. These findings provide valuable information for manufacturers to improve food spoilage detection and prevention of chilled desserts made with milk and egg.

Carcinoembryonic Antigen Cell Adhesion Molecule 1 long isoform modulates malignancy of poorly differentiated colon cancer cells.

OBJECTIVE: Nearly 20%-29% of patients with colorectal cancer (CRC) succumb to liver or lung metastasis and there is a dire need for novel targets to improve the survival of patients with metastasis. The long isoform of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-L or CC1-L) is a key regulator of immune surveillance in primary CRC, but its role in metastasis remains largely unexplored. We have examined how CC1-L expression impacts on colon cancer liver metastasis. DESIGN: Murine MC38 transfected with CC1-L were evaluated in vitro for proliferation, migration and invasion, and for in vivo experimental liver metastasis. Using shRNA silencing or pharmacological inhibition, we delineated the role in liver metastasis of Chemokine (C-C motif) Ligand 2 (CCL2) and Signal Transducer and Activator of Transcription 3 (STAT3) downstream of CC1-L. We further assessed the clinical relevance of these findings in a cohort of patients with CRC. RESULTS: MC38-CC1-L-expressing cells exhibited significantly reduced in vivo liver metastasis and displayed decreased CCL2 chemokine secretion and reduced STAT3 activity. Down-modulation of CCL2 expression and pharmacological inhibition of STAT3 activity in MC38 cells led to reduced cell invasion capacity and decreased liver metastasis. The clinical relevance of our findings is illustrated by the fact that high CC1 expression in patients with CRC combined with some inflammation-regulated and STAT3-regulated genes correlate with improved 10-year survival. CONCLUSIONS: CC1-L regulates inflammation and STAT3 signalling and contributes to the maintenance of a less-invasive CRC metastatic phenotype of poorly differentiated carcinomas.

Exploring the prognostic significance of arm-level copy number alterations in triple-negative breast cancer.

Somatic copy number alterations (SCNAs) are prevalent in cancer and play a significant role in both tumorigenesis and therapeutic resistance. While focal SCNAs have been extensively studied, the impact of larger arm-level SCNAs remains poorly understood. Here, we investigated the association between arm-level SCNAs and overall survival in triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer lacking targeted therapies. We identified frequent arm-level SCNAs, including 21q gain and 7p gain, which correlated with poor overall survival in TNBC patients. Further, we identified the expression of specific genes within these SCNAs associated with survival. Notably, we found that the expression of RIPK4, a gene located on 21q, exhibited a strong correlation with poor overall survival. In functional assays, we demonstrated that targeting Ripk4 in a murine lung metastatic TNBC model significantly reduced tumor burden, improved survival, and increased CD4+ and CD8+ T cell infiltration. RIPK4 enhanced the survival of triple-negative breast cancer cells at secondary sites, thereby facilitating the formation of metastatic lesions. Our findings highlight the significance of arm-level SCNAs in breast cancer progression and identify RIPK4 as a putative driver of TNBC metastasis and immunosuppression.

Single-cell spatial landscape of immunotherapy response reveals mechanisms of CXCL13 enhanced antitumor immunity.

BACKGROUND: Immunotherapy has revolutionized clinical outcomes for patients suffering from lung cancer, yet relatively few patients sustain long-term durable responses. Recent studies have demonstrated that the tumor immune microenvironment fosters tumorous heterogeneity and mediates both disease progression and response to immune checkpoint inhibitors (ICI). As such, there is an unmet need to elucidate the spatially defined single-cell landscape of the lung cancer microenvironment to understand the mechanisms of disease progression and identify biomarkers of response to ICI. METHODS: Here, in this study, we applied imaging mass cytometry to characterize the tumor and immunological landscape of immunotherapy response in non-small cell lung cancer by describing activated cell states, cellular interactions and neighborhoods associated with improved efficacy. We functionally validated our findings using preclinical mouse models of cancer treated with anti-programmed cell death protein-1 (PD-1) immune checkpoint blockade. RESULTS: We resolved 114,524 single cells in 27 patients treated with ICI, enabling spatial resolution of immune lineages and activation states with distinct clinical outcomes. We demonstrated that CXCL13 expression is associated with ICI efficacy in patients, and that recombinant CXCL13 potentiates anti-PD-1 response in vivo in association with increased antigen experienced T cell subsets and reduced CCR2+ monocytes. DISCUSSION: Our results provide a high-resolution molecular resource and illustrate the importance of major immune lineages as well as their functional substates in understanding the role of the tumor immune microenvironment in response to ICIs.

Obesity alters monocyte developmental trajectories to enhance metastasis.

Obesity is characterized by chronic systemic inflammation and enhances cancer metastasis and mortality. Obesity promotes breast cancer metastasis to lung in a neutrophil-dependent manner; however, the upstream regulatory mechanisms of this process remain unknown. Here, we show that obesity-induced monocytes underlie neutrophil activation and breast cancer lung metastasis. Using mass cytometry, obesity favors the expansion of myeloid lineages while restricting lymphoid cells within the peripheral blood. RNA sequencing and flow cytometry revealed that obesity-associated monocytes resemble professional antigen-presenting cells due to a shift in their development and exhibit enhanced MHCII expression and CXCL2 production. Monocyte induction of the CXCL2-CXCR2 axis underlies neutrophil activation and release of neutrophil extracellular traps to promote metastasis, and enhancement of this signaling axis is observed in lung metastases from obese cancer patients. Our findings provide mechanistic insight into the relationship between obesity and cancer by broadening our understanding of the interactive role that myeloid cells play in this process.

CEACAM1: a key regulator of vascular permeability.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is an immunoglobulin-like cell surface co-receptor expressed on epithelial, hematopoietic and endothelial cells. CEACAM1 functions as an adhesion molecule, mainly binding to itself or other members of the CEA family. We and others have previously shown that CEACAM1 is crucial for in vivo vascular integrity during ischemic neo-vascularization. Here, we have deciphered the roles of CEACAM1 in normal and pathological vascularization. We have found that Ceacam1-/- mice exhibit a significant increase in basal vascular permeability related to increased basal Akt and endothelial nitric oxide synthase (eNOS) activation in primary murine lung endothelial cells (MLECs). Moreover, CEACAM1 deletion in MLECs inhibits VEGF-mediated nitric oxide (NO) production, consistent with defective VEGF-dependent in vivo permeability in Ceacam1-/- mice. In addition, Ceacam1-null mice exhibit increased permeability of tumor vasculature. Finally, we demonstrate that CEACAM1 is tyrosine-phosphorylated upon VEGF treatment in a SHP-1- and Src-dependent manner, and that the key residues of the long cytoplasmic domain of CEACAM1 are crucial for CEACAM1 phosphorylation and NO production. This data represents the first report, to our knowledge, of a functional link between CEACAM1 and the VEGFR2/Akt/eNOS-mediated vascular permeability pathway.

CEACAM1 deficiency delays important wound healing processes.

Cutaneous wound healing is a complex process that requires the coordination of many cell types to achieve proper tissue repair. Four major overlapping processes have been identified in wound healing: hemostasis, inflammation, reepithelialization and granulation tissue formation, and tissue remodeling. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a glycoprotein expressed in epithelial, endothelial, lymphoid, and myeloid cells. Given its known roles in angiogenesis, cell migration, and immune functions, we hypothesized that CEACAM1 might also be involved in cutaneous wound healing and that a number of relevant CEACAM1-positive cell types might contribute to wound healing. To evaluate the role of CEACAM1 in these processes, 6-mm-diameter skin wounds were inflicted on Ceacam1(-/-) and wild-type mice. Herein, we demonstrate that CEACAM1 deletion indeed affects wound healing in three key ways. Infiltration of F4/80(+) macrophages was decreased in Ceacam1(-/-) wounds, altering inflammatory processes. Reepithelialization in Ceacam1(-/-) wounds was delayed. Furthermore, the vascular density of the granulation tissue in Ceacam1(-/-) wounds was significantly diminished. These results confirm CEACAM1's role as an important regulator of key processes in cutaneous wound healing, although whether this works via a specific cell type or alterations in the functioning of multiple processes remains to be determined.

Endothelial deletion of PKCδ prevents VEGF inhibition and restores blood flow reperfusion in diabetic ischemic limb.

AIMS: Peripheral artery disease is a complication of diabetes leading to critical hindlimb ischemia. Diabetes-induced inhibition of VEGF actions is associated with the activation of protein kinase Cδ (PKCδ). We aim to specifically investigate the role of PKCδ in endothelial cell (EC) function and VEGF signaling. METHODS: Nondiabetic and diabetic mice, with (ec-Prkcd-/-) or without (ec-Prkcdf/f) endothelial deletion of PKCδ, underwent femoral artery ligation. Blood flow reperfusion was assessed up to 4 weeks post-surgery. Capillary density, EC apoptosis and VEGF signaling were evaluated in the ischemic muscle. Src homology region 2 domain-containing phosphatase-1 (SHP-1) phosphatase activity was assessed in vitro using primary ECs. RESULTS: Ischemic muscle of diabetic ec-Prkcdf/f mice exhibited reduced blood flow reperfusion and capillary density while apoptosis increased as compared to nondiabetic ec-Prkcdf/f mice. In contrast, blood flow reperfusion and capillary density were significantly improved in diabetic ec-Prkcd-/- mice. VEGF signaling pathway was restored in diabetic ec-Prkcd-/- mice. The deletion of PKCδ in ECs prevented diabetes-induced VEGF unresponsiveness through a reduction of SHP-1 phosphatase activity. CONCLUSIONS: Our data provide new highlights in mechanisms by which PKCδ activation in EC contributed to poor collateral vessel formation, thus, offering novel therapeutic targets to improve angiogenesis in the diabetic limb.

Neutrophil oxidative stress mediates obesity-associated vascular dysfunction and metastatic transmigration.

Metastasis is the leading cause of cancer-related deaths, and obesity is associated with increased breast cancer (BC) metastasis. Preclinical studies have shown that obese adipose tissue induces lung neutrophilia associated with enhanced BC metastasis to this site. Here we show that obesity leads to neutrophil-dependent impairment of vascular integrity through loss of endothelial adhesions, enabling cancer cell extravasation into the lung. Mechanistically, neutrophil-produced reactive oxygen species in obese mice increase neutrophil extracellular DNA traps (NETs) and weaken endothelial junctions, facilitating the influx of tumor cells from the peripheral circulation. In vivo treatment with catalase, NET inhibitors or genetic deletion of Nos2 reversed this effect in preclinical models of obesity. Imaging mass cytometry of lung metastasis samples from patients with cancer revealed an enrichment in neutrophils with low catalase levels correlating with elevated body mass index. Our data provide insights into potentially targetable mechanisms that underlie the progression of BC in the obese population.

Ablation of angiotensin type 2 receptor prevents endothelial nitric oxide synthase glutathionylation and nitration in ischaemic abductor muscle of diabetic mice.

Peripheral artery disease is a severe complication of diabetes. We have reported that the deletion of angiotensin type 2 receptor in diabetic mice promoted vascular angiogenesis in the ischaemic muscle 4 weeks following ischaemia. However, the angiotensin type 2 receptor deletion beneficial effects occurred 2 weeks post surgery suggesting that angiotensin type 2 receptor may regulate other pro-angiogenic signalling pathways during the early phases of ischaemia. Nondiabetic and diabetic angiotensin type 2 receptor-deficient mice (Agtr2-/Y) underwent femoral artery ligation after 2 months of diabetes. Blood perfusion was measured every week up to 2 weeks post surgery. Expression of vascular endothelial growth factor, vascular endothelial growth factor receptor and endothelial nitric oxide synthase expression and activity were evaluated. Blood flow reperfusion in the ischaemic muscle of diabetic Agtr2+/Y mice was recovered at 35% as compared to a 68% recovery in diabetic Agtr2-/Y mice. The expression of vascular endothelial growth factor and its receptors was diminished in diabetic Agtr2+/Y mice, an observation not seen in diabetic Agtr2-/Y mice. Interestingly, Agtr2-/Y mice were protected from diabetes-induced glutathionylation, nitration and decreased endothelial nitric oxide synthase expression, which correlated with reduced endothelial cell death and enhanced vascular density in diabetic ischaemic muscle. In conclusion, our results suggest that the deletion of angiotensin type 2 receptor promotes blood flow reperfusion in diabetes by favouring endothelial cell survival and function.

EphA2 signaling is impacted by carcinoembryonic antigen cell adhesion molecule 1-L expression in colorectal cancer liver metastasis in a cell context-dependent manner.

We have shown that carcinoembryonic antigen cell adhesion molecule 1 long isoform (CEACAM1-L) expression in MC38 metastatic colorectal cancer (CRC) cells results in liver metastasis inhibition via CCL2 and STAT3 signaling. But other molecular mechanisms orchestrating CEACAM1-L-mediated metastasis inhibition remain to be defined. We screened a panel of mouse and human CRC cells and evaluated their metastatic outcome after CEACAM1 overexpression or downregulation. An unbiased transcript profiling and a phospho-receptor tyrosine kinase screen comparing MC38 CEACAM1-L-expressing and non-expressing (CT) CRC cells revealed reduced ephrin type-A receptor 2 (EPHA2) expression and activity. An EPHA2-specific inhibitor reduced EPHA2 downstream signaling in CT cells similar to that in CEACAM1-L cells with decreased proliferation and migration. Human CRC patients exhibiting high CEACAM1 in combination with low EPHA2 expression benefited from longer time to first recurrence/metastasis compared to those with high EPHA2 expression. With the added interaction of CEACAM6, we denoted that CEACAM1 high- and EPHA2 low-expressing patient samples with lower CEACAM6 expression also exhibited a longer time to first recurrence/metastasis. In HT29 human CRC cells, down-regulation of CEACAM1 along with CEA and CEACAM6 up-regulation led to higher metastatic burden. Overall, CEACAM1-L expression in poorly differentiated CRC can inhibit liver metastasis through cell context-dependent EPHA2-mediated signaling. However, CEACAM1's role should be considered in the presence of other CEACAM family members.

Investigation of a role for lysine residues in non-structural proteins 2 and 2/3 of the hepatitis C virus for their degradation and virus assembly.

It has been demonstrated that both uncleaved, enzymitically inactive NS2/3 and cleaved NS2 proteins are rapidly degraded upon expression in cells, phenomena described to be blocked by the addition of proteasome inhibitors. As this degradation and its regulation potentially constitute an important strategy of the hepatitis C virus (HCV) to regulate the levels of its non-structural proteins, we further investigated the turnover of these proteins in relevant RNA replication systems. A lysine-mutagenesis approach was used in an effort to prevent protein degradation and determine any effect on various steps of the viral replication cycle. We show that, while NS2-lysine mutagenesis of protease-inactive NS2/3 results in a partial stabilization of this protein, the increased NS2/3 levels do not rescue the inability of NS2/3 protease inactive replicons to replicate, suggesting that uncleaved NS2/3 is unable to functionally replace NS3 in RNA replication. Furthermore, we show that the cleaved NS2 protein is rapidly degraded in several transient and stable RNA replicon systems and that NS2 from several different genotypes also has a short half-life, highlighting the potential importance of the regulation of NS2 levels for the viral life cycle. However, in contrast to uncleaved NS2/3, neither ubiquitin nor proteasomal degradation appear to be significantly involved in NS2 degradation. Finally, although NS2 lysine-to-arginine mutagenesis does not affect this protein's levels in a JFH-1 cell culture infection system, several of these residues are identified to be involved in virion assembly, further substantiating the importance of regions of this protein for production of infectious virus.