ARNT2 controls prefrontal somatostatin interneurons mediating affective empathy.

Empathy, crucial for social interaction, is impaired across various neuropsychiatric conditions. However, the genetic and neural underpinnings of empathy variability remain elusive. By combining forward genetic mapping with transcriptome analysis, we discover that aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a key driver modulating observational fear, a basic form of affective empathy. Disrupted ARNT2 expression in the anterior cingulate cortex (ACC) reduces affect sharing in mice. Specifically, selective ARNT2 ablation in somatostatin (SST)-expressing interneurons leads to decreased pyramidal cell excitability, increased spontaneous firing, aberrant Ca2+ dynamics, and disrupted theta oscillations in the ACC, resulting in reduced vicarious freezing. We further demonstrate that ARNT2-expressing SST interneurons govern affective state discrimination, uncovering a potential mechanism by which ARNT2 polymorphisms associate with emotion recognition in humans. Our findings advance our understanding of the molecular mechanism controlling empathic capacity and highlight the neural substrates underlying social affective dysfunctions in psychiatric disorders.

The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta.

BACKGROUND: We recently reported that the dopamine (DA) analogue CA140 modulates neuroinflammatory responses in lipopolysaccharide-injected wild-type (WT) mice and in 3-month-old 5xFAD mice, a model of Alzheimer's disease (AD). However, the effects of CA140 on Aß/tau pathology and synaptic/cognitive function and its molecular mechanisms of action are unknown. METHODS: To investigate the effects of CA140 on cognitive and synaptic function and AD pathology, 3-month-old WT mice or 8-month-old (aged) 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 10, 14, or 17 days. Behavioral tests, ELISA, electrophysiology, RNA sequencing, real-time PCR, Golgi staining, immunofluorescence staining, and western blotting were conducted. RESULTS: In aged 5xFAD mice, a model of AD pathology, CA140 treatment significantly reduced Aß/tau fibrillation, Aß plaque number, tau hyperphosphorylation, and neuroinflammation by inhibiting NLRP3 activation. In addition, CA140 treatment downregulated the expression of cxcl10, a marker of AD-associated reactive astrocytes (RAs), and c1qa, a marker of the interaction of RAs with disease-associated microglia (DAMs) in 5xFAD mice. CA140 treatment also suppressed the mRNA levels of s100ß and cxcl10, markers of AD-associated RAs, in primary astrocytes from 5xFAD mice. In primary microglial cells from 5xFAD mice, CA140 treatment increased the mRNA levels of markers of homeostatic microglia (cx3cr1 and p2ry12) and decreased the mRNA levels of a marker of proliferative region-associated microglia (gpnmb) and a marker of lipid-droplet-accumulating microglia (cln3). Importantly, CA140 treatment rescued scopolamine (SCO)-mediated deficits in long-term memory, dendritic spine number, and LTP impairment. In aged 5xFAD mice, these effects of CA140 treatment on cognitive/synaptic function and AD pathology were regulated by dopamine D1 receptor (DRD1)/Elk1 signaling. In primary hippocampal neurons and WT mice, CA140 treatment promoted long-term memory and dendritic spine formation via effects on DRD1/CaMKIIα and/or ERK signaling. CONCLUSIONS: Our results indicate that CA140 improves neuronal/synaptic/cognitive function and ameliorates Aß/tau pathology and neuroinflammation by modulating DRD1 signaling in primary hippocampal neurons, primary astrocytes/microglia, WT mice, and aged 5xFAD mice.

L-serine restored lysosomal failure in cells derived from patients with BPAN reducing iron accumulation with eliminating lipofuscin.

Defective mitochondria and autophagy, as well as accumulation of lipid and iron in WDR45 mutant fibroblasts, is related to beta-propeller protein-associated neurodegeneration (BPAN). In this study, we found that enlarged lysosomes in cells derived from patients with BPAN had low enzyme activity, and most of the enlarged lysosomes had an accumulation of iron and oxidized lipid. Cryo-electron tomography revealed elongated lipid accumulation, and spectrometry-based elemental analysis showed that lysosomal iron and oxygen accumulation superimposed with lipid aggregates. Lysosomal lipid aggregates superimposed with autofluorescence as free radical generator, lipofuscin. To eliminate free radical stress by iron accumulation in cells derived from patients with BPAN, we investigated the effects of the iron chelator, 2,2'-bipyridine (bipyridyl, BIP). To study whether the defects in patient-derived cells can be rescued by an iron chelator BIP, we tested whether the level of iron and reactive oxygen species (ROS) in the cells and genes related to oxidative stress were rescued BIP treatment. Although BIP treatment decreased some iron accumulation in the cytoplasm and mitochondria, the accumulation of iron in the lysosomes and levels of cellular ROS were unaffected. In addition, the change of specific RNA levels related to free radical stress in patient fibroblasts was not rescued by BIP. To alleviate free radical stress, we investigated whether l-serine can regulate abnormal structures in cells derived from patients with BPAN through the regulation of free radical stress. l-serine treatment alleviated increase of enlarged lysosomes and iron accumulation and rescued impaired lysosomal activity by reducing oxidized lipid accumulation in the lysosomes of the cells. Lamellated lipids in the lysosomes of the cells were identified as lipofuscin through correlative light and electron microscopy, and l-serine treatment reduced the increase of lipofuscin. These data suggest that l-serine reduces oxidative stress-mediated lysosomal lipid oxidation and iron accumulation by rescuing lysosomal activity.

Paradigm shift required for translational research on the brain.

Biomedical research on the brain has led to many discoveries and developments, such as understanding human consciousness and the mind and overcoming brain diseases. However, historical biomedical research on the brain has unique characteristics that differ from those of conventional biomedical research. For example, there are different scientific interpretations due to the high complexity of the brain and insufficient intercommunication between researchers of different disciplines owing to the limited conceptual and technical overlap of distinct backgrounds. Therefore, the development of biomedical research on the brain has been slower than that in other areas. Brain biomedical research has recently undergone a paradigm shift, and conducting patient-centered, large-scale brain biomedical research has become possible using emerging high-throughput analysis tools. Neuroimaging, multiomics, and artificial intelligence technology are the main drivers of this new approach, foreshadowing dramatic advances in translational research. In addition, emerging interdisciplinary cooperative studies provide insights into how unresolved questions in biomedicine can be addressed. This review presents the in-depth aspects of conventional biomedical research and discusses the future of biomedical research on the brain.

Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma.

BACKGROUND: In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. METHODS: To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. RESULTS: CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244-/- bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. CONCLUSION: Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

Angiogenesis-on-a-chip coupled with single-cell RNA sequencing reveals spatially differential activations of autophagy along angiogenic sprouts.

Several functions of autophagy associated with proliferation, differentiation, and migration of endothelial cells have been reported. Due to lack of models recapitulating angiogenic sprouting, functional heterogeneity of autophagy in endothelial cells along angiogenic sprouts remains elusive. Here, we apply an angiogenesis-on-a-chip to reconstruct 3D sprouts with clear endpoints. We perform single-cell RNA sequencing of sprouting endothelial cells from our chip to reveal high activation of autophagy in two endothelial cell populations- proliferating endothelial cells in sprout basements and stalk-like endothelial cells near sprout endpoints- and further the reciprocal expression pattern of autophagy-related genes between stalk- and tip-like endothelial cells near sprout endpoints, implying an association of autophagy with tip-stalk cell specification. Our results suggest a model describing spatially differential roles of autophagy: quality control of proliferating endothelial cells in sprout basements for sprout elongation and tip-stalk cell specification near sprout endpoints, which may change strategies for developing autophagy-based anti-angiogenic therapeutics.

Identification of MYH9 as a key regulator for synoviocyte migration and invasion through secretome profiling.

OBJECTIVES: 'Invasive pannus' is a pathological hallmark of rheumatoid arthritis (RA). This study aimed to investigate secretome profile of synovial fibroblasts of patients with RA (RA-FLSs), a major cell type comprising the invasive pannus. METHODS: Secreted proteins from RA-FLSs were first identified using liquid chromatography-tandem mass spectrometry analysis. Ultrasonography was performed for affected joints to define synovitis severity at the time of arthrocentesis. Expression levels of myosin heavy chain 9 (MYH9) in RA-FLSs and synovial tissues were determined by ELISA, western blot analysis and immunostaining. A humanised synovitis model was induced in immuno-deficient mice. RESULTS: We first identified 843 proteins secreted from RA-FLSs; 48.5% of the secretome was associated with pannus-driven pathologies. Parallel reaction monitoring analysis of the secretome facilitated discovery of 16 key proteins related to 'invasive pannus', including MYH9, in the synovial fluids, which represented synovial pathology based on ultrasonography and inflammatory activity in the joints. Particularly, MYH9, a key protein in actin-based cell motility, showed a strong correlation with fibroblastic activity in the transcriptome profile of RA synovia. Moreover, MYH9 expression was elevated in cultured RA-FLSs and RA synovium, and its secretion was induced by interleukin-1ß, tumour necrosis factor α, toll-like receptor ligation and endoplasmic reticulum stimuli. Functional experiments demonstrated that MYH9 promoted migration and invasion of RA-FLSs in vitro and in a humanised synovitis model, which was substantially inhibited by blebbistatin, a specific MYH9 inhibitor. CONCLUSIONS: This study provides a comprehensive resource of the RA-FLS-derived secretome and suggests that MYH9 represents a promising target for retarding abnormal migration and invasion of RA-FLSs.

Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells.

Chronic viral infection impairs systemic immunity in the host; however, the mechanism underlying the dysfunction of immune cells in chronic viral infection is incompletely understood. In this study, we studied the lineage differentiation of hematopoietic stem cells (HSCs) during chronic viral infection to elucidate the changes in dendritic cell (DC) differentiation and subsequent impact on T cell functionality using a chronic lymphocytic choriomeningitis virus (LCMV) infection model. We first investigated the lineage differentiation of HSCs in the bone marrow (BM) to elucidate the modulation of immune cell differentiation and found that the populations highly restrained in their differentiation were common myeloid progenitors (CMPs) and common dendritic cell progenitors (CDPs). Of interest, the main immune cells infected with LCMV Clone 13 (CL13) in the BM were CD11b/c+ myeloid DCs. We next characterized CD11b+ DCs that differentiated during chronic LCMV infection. These DCs displayed a less immunogenic phenotype than DCs in naive or acutely infected mice, showing low expression of CD80 but high expression of PD-L1, B7-H4, IDO, TGF-ß, and IL-10. Consequently, these CD11b+ DCs induced less effective CD8+ T cells and more Foxp3+ regulatory T (Treg) cells. Furthermore, CD11b+ DCs generated during CL13 infection could not induce effective CD8+ T cells specific to the antigens of newly invading pathogens. Our findings demonstrate that DCs generated from the BM during chronic viral infection cannot activate fully functional effector CD8+ T cells specific to newly incoming antigens as well as persistent antigens themselves, suggesting a potential cause of the functional alterations in the T cell immune response during chronic viral infection.

Whole-genome sequencing reveals an association between small genomic deletions and an increased risk of developing Parkinson's disease.

Single-nucleotide variants (SNVs) associated with Parkinson's disease (PD) have been investigated mainly through genome-wide association studies. However, other genomic alterations, including copy number variations, remain less explored. In this study, we conducted whole-genome sequencing of primary (310 PD patients and 100 healthy individuals) and independent (100 PD patients and 100 healthy individuals) cohorts from the Korean population to identify high-resolution small genomic deletions, gains, and SNVs. Global small genomic deletions and gains were found to be associated with an increased and decreased risk of PD development, respectively. Thirty significant locus deletions were identified in PD, with most being associated with an increased PD risk in both cohorts. Small genomic deletions in clustered loci located in the GPR27 region had high enhancer signals and showed the closest association with PD. GPR27 was found to be expressed specifically in brain tissue, and GPR27 copy number loss was associated with upregulated SNCA expression and downregulated dopamine neurotransmitter pathways. Clustering of small genomic deletions on chr20 in exon 1 of the GNAS isoform was detected. In addition, we found several PD-associated SNVs, including one in the enhancer region of the TCF7L2 intron, which exhibited a cis-acting regulatory mode and an association with the beta-catenin signaling pathway. These findings provide a global, whole-genome view of PD and suggest that small genomic deletions in regulatory domains contribute to the risk of PD development.

Genetic deletion of nitric oxide synthase 2 ameliorates Parkinson's disease pathology and neuroinflammation in a transgenic mouse model of synucleinopathy.

Studies of mouse models of Alzheimer's disease (AD) have demonstrated that nitric oxide synthase 2 (NOS2) is involved in AD pathology. However, the effects of NOS2 on the pathology of Parkinson's disease (PD) are not well studied. To address this gap, we examined the impact of NOS2 on disease-associated phenotypes in a mouse model of PD. Transgenic mice carrying the A53T mutation of α-synuclein (SynA53T) and newly generated double transgenic mice with deletion of NOS2 (SynA53T/NOS2-/-) were used. Compared with SynA53T mice, the loss of nos2 decreased α-synuclein phosphorylation at serine 129 and reduced α-synuclein-induced microglial and astrocyte activation in SynA53T/NOS-/- mice. Additionally, neuroinflammation-related gene clusters in the deep mesencephalic nucleus (DpMe) were altered in SynA53T/NOS-/- mice compared with SynA53T mice. Taken together, our results suggest that deletion of nos2 alleviates α-synuclein pathology and α-synuclein-associated neuroinflammatory responses in the brain.

Epigenetic therapy reprograms M2-type tumor-associated macrophages into an M1-like phenotype by upregulating miR-7083-5p.

Reprogramming M2-type, pro-tumoral tumor-associated macrophages (TAMs) into M1-type, anti-tumoral macrophages is a key strategy in cancer therapy. In this study, we exploited epigenetic therapy using the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and the histone deacetylation inhibitor trichostatin A (TSA), to reprogram M2-type macrophages into an M1-like phenotype. Treatment of M2-type macrophages with the combination of 5-aza-dC and TSA decreased the levels of M2 macrophage cytokines while increasing those of M1 macrophage cytokines, as compared to the use of either therapy alone. Conditioned medium of M2 macrophages treated with the combination of 5-aza-dC and TSA sensitized the tumor cells to paclitaxel. Moreover, treatment with the combination inhibited tumor growth and improved anti-tumor immunity in the tumor microenvironment. Depletion of macrophages reduced the anti-tumor growth activity of the combination therapy. Profiling of miRNAs revealed that the expression of miR-7083-5p was remarkably upregulated in M2 macrophages, following treatment with 5-aza-dC and TSA. Transfection of miR-7083-5p reprogrammed the M2-type macrophages towards an M1-like phenotype, and adoptive transfer of M2 macrophages pre-treated with miR-7083-5p into mice inhibited tumor growth. miR-7083-5p inhibited the expression of colony-stimulating factor 2 receptor alpha and CD43 as candidate targets. These results show that epigenetic therapy upon treatment with the combination of 5-aza-dC and TSA skews M2-type TAMs towards the M1-like phenotype by upregulating miR-7083-5p, which contributes to the inhibition of tumor growth.

A systematic exploration reveals the potential of spermidine for hypopigmentation treatment through the stabilization of melanogenesis-associated proteins.

Spermidine (SPD), a polyamine naturally present in living organisms, is known to prolong the lifespan of animals. In this study, the role of SPD in melanogenesis was investigated, showing potential as a pigmenting agent. SPD treatment increased melanin production in melanocytes in a dose dependent manner. Computational analysis with RNA-sequencing data revealed the alteration of protein degradation by SPD treatment without changes in the expressions of melanogenesis-related genes. Indeed, SPD treatment significantly increased the stabilities of tyrosinase-related protein (TRP)-1 and -2 while inhibiting ubiquitination, which was confirmed by treatment of proteasome inhibitor MG132. Inhibition of protein synthesis by cycloheximide (CHX) showed that SPD treatment increased the resistance of TRP-1 and TRP-2 to protein degradation. To identify the proteins involved in SPD transportation in melanocytes, the expression of several solute carrier (SLC) membrane transporters was assessed and, among 27 transporter genes, SLC3A2, SLC7A1, SLC18B1, and SLC22A18 were highly expressed, implying they are putative SPD transporters in melanocytes. Furthermore, SLC7A1 and SLC22A18 were downregulated by SPD treatment, indicating their active involvement in polyamine homeostasis. Finally, we applied SPD to a human skin equivalent and observed elevated melanin production. Our results identify SPD as a potential natural product to alleviate hypopigmentation.

Clioquinol as an inhibitor of JmjC-histone demethylase exhibits common and unique histone methylome and transcriptome between clioquinol and hypoxia.

Clioquinol (CQ) is a hypoxic mimicker to activate hypoxia-inducible factor-1α (HIF-1α) by inhibiting HIF-1α specific asparaginyl hypoxylase (FIH-1). The structural similarity of the Jumonji C (JmjC) domain between FIH-1 and JmjC domain-containing histone lysine demethylases (JmjC-KDMs) led us to investigate whether CQ could inhibit the catalytic activities of JmjC-KDMs. Herein, we showed that CQ inhibits KDM4A/C, KDM5A/B, and KDM6B and affects H3K4me3, H3K9me3, and H3K27me3 marks, respectively. An integrative analysis of the histone methylome and transcriptome data revealed that CQ-mediated JmjC-KDM inhibition altered the transcription of target genes through differential combinations of KDMs and transcription factors. Notably, functional enrichment of target genes showed that CQ and hypoxia commonly affected the response to hypoxia, VEGF signaling, and glycolysis, whereas CQ uniquely altered apoptosis/autophagy and cytoskeleton/extracellular matrix organization. Our results suggest that CQ can be used as a JmjC-KDM inhibitor, HIF-α activator, and an alternative therapeutic agent in hypoxia-based diseases.

Nicotinamide mononucleotide reduces melanin production in aged melanocytes by inhibiting cAMP/Wnt signaling.

BACKGROUND: Nicotinamide mononucleotide (NMN) is a representative anti-aging drug that, after long-term administration in mice, causes an increase in energy and lipid metabolism, improves eye function, immune response, and increases insulin sensitivity. However, the effects of NMN on skin pigmentation are still unknown. OBJECTIVE: In this study, we aimed to demonstrate the effects of NMN on melanogenesis. METHODS: NMN was applied to both young and aged melanocytes, and melanin production, protein expression, and mRNA levels were analyzed. A reconstituted human skin model was used to validate the effect of NMN on melanogenesis in vivo. RESULTS: NMN treatment showed no apparent effects on young melanocytes, however, in aged melanocytes, a marked reduction in melanin production was observed. NMN treatment also efficiently reduced melanin production in a reconstituted human skin with aged melanocytes. Genome-wide analysis showed the downregulation of melanogenesis-related cyclic adenosine monophosphate (cAMP)/Wnt signaling in aged melanocytes. Moreover, NMN treatment downregulated forskolin-induced expression of melanogenesis-related proteins, tyrosinase (TYR), tyrosinase-related protein (TRP)- 1, and TRP-2. Nicotinamide adenine dinucleotide (NAD+), an NMN product within the cells, also reduced cAMP/Wnt signaling in aged melanocytes. SLC12A6 was the most highly expressed gene among the SLC12A family members in melanocytes and was significantly influenced by NMN or NAD+ treatment, indicating that SLC12A6 protein is an NMN transporter in melanocytes. CONCLUSION: NMN reduces melanogenesis in aged melanocytes by downregulating the signaling of melanogenesis-associated receptors. Therefore, NMN is a human-friendly anti-melanogenic agent with the potential to aid in aging-related hyperpigmentation therapy.

SENP2 suppresses browning of white adipose tissues by de-conjugating SUMO from C/EBPß.

The adipose tissue is a key site regulating energy metabolism. One of the contributing factors behind this is browning of white adipose tissue (WAT). However, knowledge of the intracellular determinants of the browning process remains incomplete. By generating adipocyte-specific Senp2 knockout (Senp2-aKO) mice, here we show that SENP2 negatively regulates browning by de-conjugating small ubiquitin-like modifiers from C/EBPß. Senp2-aKO mice are resistant to diet-induced obesity due to increased energy expenditure and heat production. Senp2 knockout promotes beige adipocyte accumulation in inguinal WAT by upregulation of thermogenic gene expression. In addition, SENP2 knockdown promotes thermogenic adipocyte differentiation of precursor cells isolated from inguinal and epididymal WATs. Mechanistically, sumoylated C/EBPß, a target of SENP2, suppresses expression of HOXC10, a browning inhibitor, by recruiting a transcriptional repressor DAXX. These findings indicate that a SENP2-C/EBPß-HOXC10 axis operates for the control of beige adipogenesis in inguinal WAT.

The macrophage odorant receptor Olfr78 mediates the lactate-induced M2 phenotype of tumor-associated macrophages.

Expression and function of odorant receptors (ORs), which account for more than 50% of G protein-coupled receptors, are being increasingly reported in nonolfactory sites. However, ORs that can be targeted by drugs to treat diseases remain poorly identified. Tumor-derived lactate plays a crucial role in multiple signaling pathways leading to generation of tumor-associated macrophages (TAMs). In this study, we hypothesized that the macrophage OR Olfr78 functions as a lactate sensor and shapes the macrophage-tumor axis. Using Olfr78+/+ and Olfr78-/- bone marrow-derived macrophages with or without exogenous Olfr78 expression, we demonstrated that Olfr78 sensed tumor-derived lactate, which was the main factor in tumor-conditioned media responsible for generation of protumoral M2-TAMs. Olfr78 functioned together with Gpr132 to mediate lactate-induced generation of protumoral M2-TAMs. In addition, syngeneic Olfr78-deficient mice exhibited reduced tumor progression and metastasis together with an increased anti- versus protumoral immune cell population. We propose that the Olfr78-lactate interaction is a therapeutic target to reduce and prevent tumor progression and metastasis.

Overexpression of PVR and PD-L1 and its association with prognosis in surgically resected squamous cell lung carcinoma.

Targeting T-Cell Immunoreceptor with Ig and ITIM domain-poliovirus receptor (PVR) pathway is a potential therapeutic strategy in lung cancer. We analyzed the expression of PVR and programmed death ligand-1 (PD-L1) in surgically resected squamous cell lung carcinoma (SQCC) and determined its prognostic significance. We collected archival surgical specimens and data of 259 patients with SQCC at Yonsei Cancer Center (1998-2020). Analysis of variance was used to analyze the correlations between PVR and PD-L1 expression and patient characteristics. Kaplan-Meier curves were used to estimate recurrence-free survival (RFS) and overall survival (OS). Most patients were male (93%); the majority were diagnosed with stage 1 (47%), followed by stage 2 (29%) and stage 3 (21%). Overexpression of PVR resulted in a significantly shorter median RFS and OS (P = 0.01). PD-L1 expression was not significant in terms of prognosis. Patients were subdivided into four groups based on low and high PVR and PD-L1 expression. Those expressing high levels of PVR and PD-L1 had the shortest RFS (P = 0.03). PVR overexpression is associated with a poor prognosis in surgically resected SQCC. Inhibition of PVR as well as PD-L1 may help overcome the lack of response to immune checkpoint monotherapy.

Hypoxia-Driven HIF-1α Activation Reprograms Pre-Activated NK Cells towards Highly Potent Effector Phenotypes via ERK/STAT3 Pathways.

NK cells are the predominant innate lymphocyte subsets specialized to kill malignant tumor cells. In patients with advanced cancer, hypoxic stress shapes NK cells toward tumor-resistant and immunosuppressive phenotypes, hence a strategy to restore NK function is critical for successful tumor immunotherapy. Here, we present evidence that pre-activation and subsequent HIF-1α-dependent metabolic shift of NK cells from oxidative phosphorylation into glycolysis are keys to overcome hypoxia-mediated impairment in NK cell survival, proliferation, and tumor cytotoxicity. Specifically, exposing NK cells to 7-9 days of normoxic culture followed by a pO2 of 1.5% hypoxia led to a highly potent effector phenotype via HIF-1α stabilization and upregulation of its target genes, BNIP3, PDK1, VEGF, PKM2, and LDHA. RNA sequencing and network analyses revealed that concomitant reduction of p21/p53 apoptotic pathways along with upregulation of cell cycle-promoting genes, CCNE1, CDC6, CDC20, and downregulation of cell cycle-arrest genes, CDKN1A, GADD45A, and MDM2 were accountable for superior expansion of NK cells via ERK/STAT3 activation. Furthermore, HIF-1α-dependent upregulation of the NKp44 receptor in hypoxia-exposed NK cells resulted in increased killing against K562, CEM, and A375 tumor targets both in-vitro and in-vivo tumor clearance assays. Therefore, hypoxic exposure on pre-activated proliferating NK cells triggered HIF-1α-dependent pathways to initiate coordinated regulation of cell cycle, apoptosis, and cytotoxicity at the global gene transcription level. Our results uncover a previously unidentified role of HIF-1α-mediated metabolic reprogramming that can reverse impaired NK effector phenotypes to generate requisite numbers of functionally robust NK cells for adoptive cellular therapy for clinical evaluation.