An autophagy program that promotes T cell egress from the lymph node controls responses to immune checkpoint blockade.

Lymphatic endothelial cells (LECs) of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady-state and tumor-bearing conditions is lacking. Intravital imaging combined with immune phenotyping shows that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs by increasing the availability of the main egress signal sphingosine-1-phosphate. Single-cell RNA sequencing of tumor-draining LNs shows that loss of ATG5 remodels niche-specific LEC phenotypes involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC autophagy prevents recruitment of tumor-infiltrating T and natural killer cells and abrogates response to immunotherapy. Thus, an LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics.

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping.

Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.

Tumor endothelial cell autophagy is a key vascular-immune checkpoint in melanoma.

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.

Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma.

Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells.

Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis.

Autophagy has vasculoprotective roles, but whether and how it regulates lymphatic endothelial cells (LEC) homeostasis and lymphangiogenesis is unknown. Here, we show that genetic deficiency of autophagy in LEC impairs responses to VEGF-C and injury-driven corneal lymphangiogenesis. Autophagy loss in LEC compromises the expression of main effectors of LEC identity, like VEGFR3, affects mitochondrial dynamics and causes an accumulation of lipid droplets (LDs) in vitro and in vivo. When lipophagy is impaired, mitochondrial ATP production, fatty acid oxidation, acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes are dwindled. Enforcing mitochondria fusion by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC fails to restore LDs turnover and lymphatic gene expression, whereas supplementing the fatty acid precursor acetate rescues VEGFR3 levels and signaling, and lymphangiogenesis in LEC-Atg5-/- mice. Our findings reveal that lipophagy in LEC by supporting FAO, preserves a mitochondrial-PROX1 gene expression circuit that safeguards LEC responsiveness to lymphangiogenic mediators and lymphangiogenesis.

The glycoprotein GP130 governs the surface presentation of the G protein-coupled receptor APLNR.

Glioblastoma is one of the most lethal forms of adult cancer, with a median survival of ∼15 mo. Targeting glioblastoma stem-like cells (GSCs) at the origin of tumor formation and relapse may prove beneficial. In situ, GSCs are nested within the vascular bed in tight interaction with brain endothelial cells, which positively control their expansion. Because GSCs are notably addicted to apelin (APLN), sourced from the surrounding endothelial stroma, the APLN/APLNR nexus has emerged as a druggable network. However, how this signaling axis operates in gliomagenesis remains underestimated. Here, we find that the glycoprotein GP130 interacts with APLNR at the plasma membrane of GSCs and arbitrates its availability at the surface via ELMOD1, which may further impact on ARF-mediated endovesicular trafficking. From a functional standpoint, interfering with GP130 thwarts APLNR-mediated self-renewal of GSCs ex vivo. Thus, GP130 emerges as an unexpected cicerone to the G protein-coupled APLN receptor, opening new therapeutic perspectives toward the targeting of cancer stem cells.

Lysosomes in glioblastoma: pump up the volume.

Lysosomes are acidic, dynamic organelles that supervise catabolism, integrate signaling cascades, and tune cellular trafficking. Moreover, the loss of their integrity may jeopardize cell viability. In cancer cells, lysosomes are qualitatively and quantitatively modified for the tumor's own benefit. For all these reasons, these organelles emerge as appealing intracellular targets to manipulate non-oncogene addiction. This is of particular interest for brain diseases, including neurodegenerative disorders and cancer, in which stem cells are exhausted and transformed, respectively. Recent publications had demonstrated that stem cells displayed disarmed lysosomes in terms of number and functions during aging and oncogenic progression. Likewise, our laboratory identified that the arginine protease MALT1, normally dedicated to the assembly of proper NF-kB activation and processing a number of substrates, arbitrates lysosome biogenesis and mTOR signaling in glioblastoma stem-like cells. Indeed, blocking either the expression or the activity of this enzyme leads to an aberrant increase of lysosomes, alongside of the down-regulation of the mTOR signaling. This surge of lysosomes eradicates glioblastoma stem-like cells. Targeting lysosomes might thus inspire the design of new strategies to face this devastating human cancer. Here, we provide an overview of the functions of the lysosome as well as its role as a cell death initiator, to highlight the potential of lysosomal drugs for glioblastoma therapy.

Paracaspase MALT1 regulates glioma cell survival by controlling endo-lysosome homeostasis.

Glioblastoma is one of the most lethal forms of adult cancer with a median survival of around 15 months. A potential treatment strategy involves targeting glioblastoma stem-like cells (GSC), which constitute a cell autonomous reservoir of aberrant cells able to initiate, maintain, and repopulate the tumor mass. Here, we report that the expression of the paracaspase mucosa-associated lymphoid tissue l (MALT1), a protease previously linked to antigen receptor-mediated NF-κB activation and B-cell lymphoma survival, inversely correlates with patient probability of survival. The knockdown of MALT1 largely impaired the expansion of patient-derived stem-like cells in vitro, and this could be recapitulated with pharmacological inhibitors, in vitro and in vivo. Blocking MALT1 protease activity increases the endo-lysosome abundance, impairs autophagic flux, and culminates in lysosomal-mediated cell death, concomitantly with mTOR inactivation and dispersion from endo-lysosomes. These findings place MALT1 as a new druggable target involved in glioblastoma and unveil ways to modulate the homeostasis of endo-lysosomes.

Information Priorities for Deciding on Treatment of Pelvic Organ Prolapse.

OBJECTIVE: The objective of this study was to elicit information priorities from women considering treatment for pelvic organ prolapse (POP). STUDY DESIGN: This is a cross-sectional study of women before and after treatment of stage II or higher POP. Women were recruited either at the conclusion of their initial evaluation (before treatment) or at postoperative or pessary maintenance visits (after treatment). Women completed a written survey that used a Likert scale to rank potentially frequently asked questions (FAQs) that could be important information to use in decision making for POP. RESULTS: Among the 100 women surveyed, 32 women wanted to pursue surgical options and 18 women wanted to pursue nonsurgical treatment options in the before treatment group. In the after treatment group, 35 women had undergone surgery and 15 women were using a pessary.Overall, women ranked FAQs about treatment success (overall Likert score, 1.11±0.35), quality of life after treatment (1.18±0.41), and complications and side effects (1.20±0.57) as the most important information when making a decision. Women were least concerned with FAQs regarding cost (2.39±1.48), impact on sexual function (2.21±1.4), and impact on hormones (2.20±1.27). CONCLUSIONS: Women with POP identified the most important FAQs related to treatment success and complications, quality of life, and understanding how the treatment works. This information will be used to develop a comprehensive decision aid for women considering treatment options for POP.

3D Endothelial Cell Migration.

Endothelial cells have the capacity to shift between states of quiescence and angiogenesis. The early stage of angiogenesis, sprouting, occurs with the synchronized activities of tip cells, which lead the migration of the sprout, and stalk cells, which elongate this vessel sprout. Here, we describe a method to study in vitro this early and rapid stage of sprouting angiogenesis.

Pharmacological targeting of apelin impairs glioblastoma growth.

Glioblastoma are highly aggressive brain tumours that are associated with an extremely poor prognosis. Within these tumours exists a subpopulation of highly plastic self-renewing cancer cells that retain the ability to expand ex vivo as tumourspheres, induce tumour growth in mice, and have been implicated in radio- and chemo-resistance. Although their identity and fate are regulated by external cues emanating from endothelial cells, the nature of such signals remains unknown. Here, we used a mass spectrometry proteomic approach to characterize the factors released by brain endothelial cells. We report the identification of the vasoactive peptide apelin as a central regulator for endothelial-mediated maintenance of glioblastoma patient-derived cells with stem-like properties. Genetic and pharmacological targeting of apelin cognate receptor abrogates apelin- and endothelial-mediated expansion of glioblastoma patient-derived cells with stem-like properties in vitro and suppresses tumour growth in vivo. Functionally, selective competitive antagonists of apelin receptor were shown to be safe and effective in reducing tumour expansion and lengthening the survival of intracranially xenografted mice. Therefore, the apelin/apelin receptor signalling nexus may operate as a paracrine signal that sustains tumour cell expansion and progression, suggesting that apelin is a druggable factor in glioblastoma.

Role of WNT7B-induced noncanonical pathway in advanced prostate cancer.

Advanced prostate cancer is characterized by incurable castration-resistant progression and osteoblastic bone metastasis. While androgen deprivation therapy remains the primary treatment for advanced prostate cancer, resistance inevitably develops. Importantly, mounting evidence indicates that androgen receptor (AR) signaling continues to play a critical role in the growth of advanced prostate cancer despite androgen deprivation. While the mechanisms of aberrant AR activation in advanced prostate cancer have been extensively studied, the downstream AR target genes involved in the progression of castration resistance are largely unknown. Here, we identify WNT7B as a direct AR target gene highly expressed in castration-resistant prostate cancer (CRPC) cells. Our results show that expression of WNT7B is necessary for the growth of prostate cancer cells and that this effect is enhanced under androgen-deprived conditions. Further analyses reveal that WNT7B promotes androgen-independent growth of CRPC cells likely through the activation of protein kinase C isozymes. Our results also show that prostate cancer-produced WNT7B induces osteoblast differentiation in vitro through a direct cell-cell interaction, and that WNT7B is upregulated in human prostate cancer xenografts that cause an osteoblastic reaction when grown in bone. Taken together, these results suggest that AR-regulated WNT7B signaling is critical for the growth of CRPC and development of the osteoblastic bone response characteristic of advanced prostate cancer.