Development of a clinical prediction score for perioperative complications following metastatic spinal surgery (PERCOM) score.

Background: Spinal metastases can impair mobility, worsening the Karnofsky Performance Status (KPS). Surgery for spinal metastases has the potential to improve KPS and extend prognosis, but it is crucial to recognize the elevated risk of perioperative complications. Therefore, the development of a new scoring system to accurately predict perioperative complications in spinal metastatic surgery is essential. Methods: We conducted a retrospective observational study with 86 patients who underwent surgical intervention for spinal metastases. Patients were divided into two groups based on the presence or absence of perioperative complications within 14 days after surgery. Various factors related to perioperative complications were assessed through univariate and multivariate analyses. We established a clinical prognostic scoring system called the Perioperative Complications following Metastatic Spinal Surgery (PERCOM) score and evaluated its precision using receiver operating characteristic (ROC) analysis. Results: Five variables (age, KPS, primary prostate cancer, Albumin, and Hemoglobin) identified in the univariate analysis were assigned binary values of 0 or 1. The PERCOM score was then calculated for each patient by summing the individual points, ranging from 0 to 5. The optimal threshold determined by ROC curve analysis for the PERCOM score was 2 points, with a sensitivity of 86 % and a specificity of 56 %. Conclusions: The composite PERCOM score effectively predicted perioperative complications in spinal metastasis surgery. To further validate its precision, a prospective multicenter study is needed.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell-cell interactions in metastatic niches.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

PDIVAS: Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing.

BACKGROUND: Deep-intronic variants that alter RNA splicing were ineffectively evaluated in the search for the cause of genetic diseases. Determination of such pathogenic variants from a vast number of deep-intronic variants (approximately 1,500,000 variants per individual) represents a technical challenge to researchers. Thus, we developed a Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing (PDIVAS) to easily detect pathogenic deep-intronic variants. RESULTS: PDIVAS was trained on an ensemble machine-learning algorithm to classify pathogenic and benign variants in a curated dataset. The dataset consists of manually curated pathogenic splice-altering variants (SAVs) and commonly observed benign variants within deep introns. Splicing features and a splicing constraint metric were used to maximize the predictive sensitivity and specificity, respectively. PDIVAS showed an average precision of 0.92 and a maximum MCC of 0.88 in classifying these variants, which were the best of the previous predictors. When PDIVAS was applied to genome sequencing analysis on a threshold with 95% sensitivity for reported pathogenic SAVs, an average of 27 pathogenic candidates were extracted per individual. Furthermore, the causative variants in simulated patient genomes were more efficiently prioritized than the previous predictors. CONCLUSION: Incorporating PDIVAS into variant interpretation pipelines will enable efficient detection of disease-causing deep-intronic SAVs and contribute to improving the diagnostic yield. PDIVAS is publicly available at https://github.com/shiro-kur/PDIVAS .

CCL21-Ser expression in melanoma cells recruits CCR7+ naïve T cells to tumor tissues and promotes tumor growth.

CCL21-Ser, a chemokine encoded by the Ccl21a gene, is constitutively expressed in the thymic epithelial cells and stromal cells of secondary lymphoid organs. It regulates immune cell migration and survival through its receptor CCR7. Herein, using CCL21-Ser-expressing melanoma cells and the Ccl21a-deficient mice, we demonstrated the functional role of cancer cell-derived CCL21-Ser in melanoma growth in vivo. The B16-F10 tumor growth was significantly decreased in Ccl21a-deficient mice compared with that in wild-type mice, indicating that host-derived CCL21-Ser contributes to melanoma proliferation in vivo. In Ccl21a-deficient mice, tumor growth of melanoma cells expressing CCL21-Ser was significantly enhanced, suggesting that CCL21-Ser from melanoma cells promotes tumor growth in the absence of host-derived CCL21-Ser. The increase in tumor growth was associated with an increase in the CCR7+ CD62L+ T cell frequency in the tumor tissue but was inversely correlated with Treg frequency, suggesting that naïve T cells primarily promote tumor growth. Adoptive transfer experiments demonstrated that naïve T cells are preferentially recruited from the blood into tumors with melanoma cell-derived CCL21-Ser expression. These results suggest that CCL21-Ser from melanoma cells promotes the infiltration of CCR7+ naïve T cells into the tumor tissues and creates a tumor microenvironment favorable for melanoma growth.

Integrated-gut-liver-on-a-chip platform as an in vitro human model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) afflicts a significant percentage of the population; however, no effective treatments have yet been established because of the unsuitability of in vitro assays and animal experimental models. Here, we present an integrated-gut-liver-on-a-chip (iGLC) platform as an in vitro human model of the gut-liver axis (GLA) by co-culturing human gut and liver cell lines interconnected via microfluidics in a closed circulation loop, for the initiation and progression of NAFLD by treatment with free fatty acids (FFAs) for 1 and 7 days, respectively. Co-cultured Caco-2 gut-mimicking cells and HepG2 hepatocyte-like cells demonstrate the protective effects from apoptosis against FFAs treatment, whereas mono-cultured cells exhibit induced apoptosis. Phenotype and gene expression analyses reveal that the FFAs-treated gut and liver cells accumulated intracellular lipid droplets and show an increase in gene expression associated with a cellular response to copper ions and endoplasmic reticulum stress. As an in vitro human GLA model, the iGLC platform may serve as an alternative to animal experiments for investigating the mechanisms of NAFLD.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism by which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3 -/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8 -/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8 -/ bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism in which migrating macrophages attracted astrocytes and affected the pathophysiology and outcome after SCI.

Identification of the α2 chain of interleukin-13 receptor as a potential biomarker for predicting castration resistance of prostate cancer using patient-derived xenograft models.

BACKGROUND: Several treatment strategies use upfront chemotherapy or androgen receptor axis-targeting therapies for metastatic prostate cancer. However, there are no useful biomarkers for selecting appropriate patients who urgently require these treatments. METHODS: Novel patient-derived xenograft (PDX) castration-sensitive and -resistant models were established and gene expression patterns were comprehensively compared. The function of a gene highly expressed in the castration-resistant models was evaluated by its overexpression in LNCaP prostate cancer cells. Protein expression in the tumors and serum of patients was examined by immunohistochemistry and ELISA, and correlations with castration resistance were analyzed. RESULTS: Expression of the α2 chain of interleukin-13 receptor (IL13Rα2) was higher in castration-resistant PDX tumors. LNCaP cells overexpressing IL13Rα2 acquired castration resistance in vitro and in vivo. In tissue samples, IL13Rα2 expression levels were significantly associated with castration-resistant progression (p < 0.05). In serum samples, IL13Rα2 levels could be measured in 5 of 28 (18%) castration-resistant prostate cancer patients. CONCLUSION: IL13Rα2 was highly expressed in castration-resistant prostate cancer PDX models and was associated with the castration resistance of prostate cancer cells. It might be a potential tissue and serum biomarker for predicting castration resistance in prostate cancer patients.

Glial scar survives until the chronic phase by recruiting scar-forming astrocytes after spinal cord injury.

Spinal cord injury (SCI) causes reactive astrogliosis, the sequential phenotypic change of astrocytes in which naïve astrocytes (NAs) transform into reactive astrocytes (RAs) and subsequently become scar-forming astrocytes (SAs), resulting in glial scar formation around the lesion site and thereby limiting axonal regeneration and motor/sensory functional recovery. Inhibiting the transformation of RAs into SAs in the acute phase attenuates the reactive astrogliosis and promotes regeneration. However, whether or not SAs once formed can revert to RAs or SAs is unclear. We performed selective isolation of astrocytes from glial scars at different time points for a gene expression analysis and found that the expression of Sox9, an important transcriptional factor for glial cell differentiation, was significantly increased in chronic phase astrocytes (CAs) compared to SAs in the sub-acute phase. Furthermore, CAs showed a significantly lower expression of chondroitin sulfate proteoglycan (CSPG)-related genes than SAs. These results indicated that SAs changed their phenotypes according to the surrounding environment of the injured spinal cord over time. Even though the integrin-N-cadherin pathway is critical for glial scar formation, collagen-I-grown scar-forming astrocytes (Col-I-SAs) did not change their phenotype after depleting the effect of integrin or N-cadherin. In addition, we found that Col-I-SAs transplanted into a naïve spinal cord formed glial scar again by maintaining a high expression of genes involved in the integrin-N-cadherin pathway and a low expression of CSPG-related genes. Interestingly, the transplanted Col-I-SAs changed NAs into SAs, and anti-ß1-integrin antibody blocked the recruitment of SAs while reducing the volume of glial scar in the chronic phase. Our findings indicate that while the characteristics of glial scars change over time after SCI, SAs have a cell-autonomous function to form and maintain a glial scar, highlighting the basic mechanism underlying the persistence of glial scars after central nervous system injury until the chronic phase, which may be a therapeutic target.

Chemical induction of splice-neoantigens attenuates tumor growth in a preclinical model of colorectal cancer.

Neoantigen production is a determinant of cancer immunotherapy. However, the expansion of neoantigen abundance for cancer therapeutics is technically challenging. Here, we report that the synthetic compound RECTAS can induce the production of splice-neoantigens that could be used to boost antitumor immune responses. RECTAS suppressed tumor growth in a CD8+ T cell- and tumor major histocompatibility complex class I-dependent manner and enhanced immune checkpoint blockade efficacy. Subsequent transcriptome analysis and validation for immunogenicity identified six splice-neoantigen candidates whose expression was induced by RECTAS treatment. Vaccination of the identified neoepitopes elicited T cell responses capable of killing cancer cells in vitro, in addition to suppression of tumor growth in vivo upon sensitization with RECTAS. Collectively, these results provide support for the further development of splice variant-inducing treatments for cancer immunotherapy.

Method for Identifying Sequence Motifs in Pre-miRNAs for Small-Molecule Binding.

Non-coding RNAs are emerging targets for drug development because they are involved in various cellular processes. However, there are a few reliable design strategies for small molecules that can target RNAs. This paper reports a simple and efficient method to comprehensively analyze RNA motifs that can be bound by a specific small molecule. The method involves Dicer-mediated pre-miRNA cleavage and subsequent analysis of the reaction products by high-throughput sequencing. A pre-miRNA mutant library containing a randomized region at the Dicer cleavage site was used as the substrate for the reaction. Sequencing analysis of the products of the reaction carried out in the presence or absence of a synthetic small molecule identified the pre-miRNA mutants whose Dicer-mediated cleavage was significantly altered by the addition of the small molecule. The binding of the small molecule to the identified pre-miRNA mutants was confirmed by surface plasmon resonance, demonstrating the feasibility of our method.

Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage.

We have previously shown that Kyoto University Substances (KUSs), valosin-containing protein (VCP) modulators, suppress cell death in retinal ganglion cells of glaucoma mouse models through alterations of various genes expressions. In this study, among the genes whose expression in retinal ganglion cells was altered by KUS treatment in the N-methyl-D-aspartic acid (NMDA) injury model, we focused on two genes, endothelin-1 (Edn1) and endothelin receptor type B (Ednrb), whose expression was up-regulated by NMDA and down-regulated by KUS treatment. First, we confirmed that the expression of Edn1 and Ednrb was upregulated by NMDA and suppressed by KUS administration in mice retinae. Next, to clarify the influence of KUSs on cell viability in relation to the endothelin signaling, cell viability was examined with or without antagonists or agonists of endothelin and with or without KUS in 661W retinal cells under stress conditions. KUS showed a significant protective effect under glucose-free conditions and tunicamycin-induced stress. This protective effect was partially attenuated in the presence of an endothelin antagonist or agonist under glucose-free conditions. These results suggest that KUSs protect cells partially by suppressing the upregulated endothelin signaling under stress conditions.

Therapeutic manipulation of IKBKAP mis-splicing with a small molecule to cure familial dysautonomia.

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5' splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.

CDC2-like (CLK) protein kinase inhibition as a novel targeted therapeutic strategy in prostate cancer.

Dysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE, ESCO2, CKAP2, MELK, ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer.

Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region.

Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) is widely used in research and clinical diagnosis. Improvement of MMLV RT thermostability has been an important topic of research for increasing the efficiency of cDNA synthesis. In this study, we attempted to increase MMLV RT thermostability by introducing a disulfide bridge in its RNase H region using site-directed mutagenesis. Five variants were designed, focusing on the distance between the two residues to be mutated into cysteine. The variants were expressed in Escherichia coli and purified. A551C/T662C was determined to be the most thermostable variant.

S1PR3-G12-biased agonist ALESIA targets cancer metabolism and promotes glucose starvation.

Metabolic activities are altered in cancer cells compared with those in normal cells, and the cancer-specific pathway becomes a potential therapeutic target. Higher cellular glucose consumption, which leads to lower glucose levels, is a hallmark of cancer cells. In an objective screening for chemicals that induce cell death under low-glucose conditions, we discovered a compound, denoted as ALESIA (Anticancer Ligand Enhancing Starvation-induced Apoptosis). By our shedding assay of transforming growth factor α in HEK293A cells, ALESIA was determined to act as a sphingosine-1-phosphate receptor 3-G12-biased agonist that promotes nitric oxide production and oxidative stress. The oxidative stress triggered by ALESIA resulted in the exhaustion of glucose, cellular NADPH deficiency, and then cancer cell death. Intraperitoneal administration of ALESIA improved the survival of mice with peritoneally disseminated rhabdomyosarcoma, indicating its potential as a new type of anticancer drug for glucose starvation therapy.

Examining the Beneficial Aspects of Nutritional Guidance Using Estimated Daily Salt Intake in Cancer Patients with Ischemic Heart Disease.

BACKGROUND The outcomes associated with nutritional guidance for patients with ischemic heart disease undergoing cancer treatment have not been explored. We examined the effects of nutritional guidance using estimated daily salt intake in cancer patients with ischemic heart disease. MATERIAL AND METHODS We examined the data from physical examinations and laboratory assessments of 27 patients with suspected excessive salt intake who underwent coronary angiography for the first time and received nutritional guidance on their next visit to the Department of Cardiology of Shizuoka Cancer Center between May 2018 and March 2020. Salinity measurement was not used in the nutritional guidance method, but the patients were instructed to reduce consumption of salt-containing foods. We compared the frequency of the estimated daily salt intake with the frequency of categories requiring salt control (food, cooking, and table salts). RESULTS The median age of the participants was 74 (range, 63-86) years. The estimated daily salt intake and the rate of change in the triglyceride level were negatively correlated (r=-0.61, P<0.01). The estimated daily salt intake was reduced in 16 cases; there was a relative decrease in the frequency of food intake among categories requiring salt control compared with that in the nonimproved cases (P<0.01). No difference was found between the cancer stage and the affected site of the digestive system in either group (P=0.64, P=0.39). CONCLUSIONS Nutritional guidance on dietary habits without using salinity measurement was beneficial in preventing ischemic heart disease and food intake reduction in cancer patients.

Long-term outcomes of spinal meningioma resection with outer layer of dura preservation technique.

Spinal meningioma is a common benign intradural spinal tumor. It has been reported that the local recurrence rate after surgical resection increases with longer follow-up duration. Simpson grade 1 resection could reduce the risk of recurrence, but this procedure needs dural reconstruction, which would cause cerebrospinal fluid (CSF) leakage or iatrogenic spinal cord injury. Saito et al. reported dura preservation technique to reduce the risk of CSF leakage, in which the meningioma together with the inner layer of the dura is removed and the outer layer is preserved for simple dural closure. The long-term outcomes with this technique have never been investigated. In this study, we retrospectively analyzed the data of 38 surgically treated patients (dura preservation technique, 12 patients; Simpson grade 2 resection, 26 patients) to assess the long-term recurrence rate (mean, 121.5 months; range, 60-228 months). The local recurrence rate in the dura preservation group was 8.3% (1 of 12 cases), which was similar to that in Simpson grade 2 resection group (2 of 26 cases [7.7%]). Although this case series did not indicate the significant difference in the recurrence rates between the dura preservation group and Simpson grade 2 group, we consider that this technique still has advantages for surgically less invasiveness in terms of dural reconstruction which is necessary for Simpson grade 1 and higher possibility of complete resection of tumors compared with Simpson grade 2 resection.

Comparison of Measured Data between Pre- and Post-Radiotherapy in a Patient with Cardiac Resynchronization Therapy Defibrillator.

Although some researches proved the influence of radiation therapy (RT) on pacemakers and implantable cardioverter defibrillators, little has been reported on cardiac resynchronization therapy defibrillators (CRTDs). We experienced a case of RT on CRTD and had a new finding.A patient with CRTD implanted for dilated cardiomyopathy was diagnosed with lung squamous cell carcinoma and started receiving RT. All the implanted devices, including the main body of CRTD, left ventricular lead (LV), right ventricular lead with high-voltage conductor, and right atrial lead, were from the same manufacturer. The radiation targeted the tumor of 67 mm in diameter in the right superior lobe for 5 min per session. The CRTD was outside the radiation field, which is 65 mm, but the leads were inside. Plan 1 used 2 Gy/fr with 8 megavolt photons, and Plan 1 was irradiated at 0° and 180° for 16 RT sessions. The dosage was increased to 3 Gy for Plan 2 for 4 sessions. Plan 3 used 2 Gy with 6 and 8 megavolt photons, and Plan 3 was irradiated at 27.7° and 200.7° for 11 RT sessions. Changes in measured parameters were assessed before and after RT.Changes in impedance of LV and high-voltage lead exceeded prespecified threshold. However, no significant errors were detected in the CRTD on the dosages and energy we used.We hypothesize that the lead insulator could have been affected by radiation.

Identification of Qk as a Glial Precursor Cell Marker that Governs the Fate Specification of Neural Stem Cells to a Glial Cell Lineage.

During brain development, neural stem cells (NSCs) initially produce neurons and change their fate to generate glias. While the regulation of neurogenesis is well characterized, specific markers for glial precursor cells (GPCs) and the master regulators for gliogenesis remain unidentified. Accumulating evidence suggests that RNA-binding proteins (RBPs) have significant roles in neuronal development and function, as they comprehensively regulate the expression of target genes in a cell-type-specific manner. We systematically investigated the expression profiles of 1,436 murine RBPs in the developing mouse brain and identified quaking (Qk) as a marker of the putative GPC population. Functional analysis of the NSC-specific Qk-null mutant mouse revealed the key role of Qk in astrocyte and oligodendrocyte generation and differentiation from NSCs. Mechanistically, Qk upregulates gliogenic genes via quaking response elements in their 3' untranslated regions. These results provide crucial directions for identifying GPCs and deciphering the regulatory mechanisms of gliogenesis from NSCs.