Expression Of Secreted Phosphoprotein Research Articles

OP24 SPP1 in colitis-associated colon cancer

Abstract Background Patients with ulcerative colitis (UC) and Crohn’s disease (CD) face a lifelong risk of developing colitis-associated carcinoma (CAC). Current insights into CAC development originate from murine CAC models, while human CAC studies primarily focus on mutational analyses. Although the mutational landscape reveals distinct patterns and frequencies compared to colorectal cancer, it falls short in elucidating the inflammatory mechanisms of CAC development. Consequently, we adopted a multi-omics approach to unravel CAC carcinogenesis, utilizing human samples from patients with CD, CD-associated CAC (CD-CAC), UC, and UC-associated CAC (UC-CAC). Findings were further validated in vitro using human colon organoids. Methods A Nanostring Immunology v2 panel was employed on RNA (archival FFPE sections) from 40 patients suffering from either IBD or CAC, alongside inflammation-free healthy controls (n=10 per group). Our data revealed a robust upregulation of the SPP1 (Secreted Phosphoprotein 1) gene encoding osteopontin (OPN) in CAC patients. Prospectively, we extended our methodology to an unbiased imaging-based spatial RNA in situ sequencing (ISS) approach (477 genes, Xenium, 10X Genomics) using FFPE sections from CAC and UC patients (n=3 per group). This provided insights into the spatial expression of SPP1 and its target receptors at a subcellular resolution. Findings were validated using whole FFPE section confocal microscopy. In addition, in vitro models, including human UC organoids, were analyzed by bulk RNA-Seq, RT-qPCR, and downstream functional assays. Results Nanostring Immunology v2 gene expression data revealed upregulation of the SPP1 gene encoding OPN (Fig 1A) and EMT transcription factors FN1 and ZEB1 in CAC patients. Xenium RNA in situ sequencing (ISS) unveiled the spatial heterogeneity of SPP1 expression in CAC (Fig 1B). Unsupervised graph-based clustering identified an SPP1+CD68+ macrophage cluster exclusively in CAC (Fig 1C), validated through OPN/CD68 immunostainings (Fig 1D). In vitro OPN stimulation across multiple human and mice colon organoid models excluded OPN as the trigger of EMT. Interestingly, Xenium ISS and immunostainings revealed a mutually exclusive spatial location of SPP1/OPN+ macrophages and CD8+ T cells (Fig 1E, F and G). Conclusion SPP1 is significantly upregulated in CAC (RNA and protein), expressed by CD68+ macrophages. In vitro OPN stimulation demonstrated no direct effect on intestinal epithelial organoids. The mutually exclusive spatial locations of SPP1/OPN+ macrophages and CD8+ T cells suggests a crucial role of SPP1/OPN in modulating the immunosurveillance of CAC cells by CD8+ tumour lymphocytes.

SPP1 mRNA determination based on molecular beacon for the recurrence prognosis of bladder cancer.

Bladder cancer (BC) has attracted significant attention on account of its recurrence as well as mortality. Tumor recurrence plays a significant role in cancer patients' individual treatment. Secreted phosphoprotein 1 (SPP1) has been recognized as a potential target for treating BC and served as a useful biomarker for prognosis; it is commonly tested by immunohistochemistry (IHC). However, this conventional method has the disadvantage of being time-consuming and costly. This study aimed to develop a molecular beacon (MB) for the detection of SPP1 messenger RNA (mRNA) for the recurrence prognosis of BC. An MB was constructed and applied to image SPP1 mRNA level at both molecular and cellular level. The fluorescence spectra were recorded with a fluorescence spectrophotometer. The effect of SPP1 MB toward the cell viability was performed by Cell Counting Kit-8 (CCK-8) assays. The SPP1 mRNA expression level was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cancer cells and tissues were analyzed with confocal fluorescence imaging. Correlation, sensitivity, and specificity parameters were calculated. It was demonstrated that both cancer cells and BC tissues expressed high signal which reflected the expression of SPP1. In addition, 42 cases were detected by MB and divided into two groups according to the fluorescence intensity. The results further suggested that highly expressed SPP1 could predict early tumor recurrence in BC. The SPP1 MB could be applied as an appropriate approach to predict BC recurrence and patients' prognosis.

Exosomes derived from mesenchymal stromal cells exert a therapeutic effect on hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway

ObjectiveHypoxic pulmonary hypertension (HPH) is a progressive and life-threatening disease characterized by perivascular inflammation, pulmonary vascular remodeling, and occlusion. Mesenchymal stromal cell-derived exosomes (MSC-exo) have emerged as potential therapeutic agents due to their role in cell communication and the transportation of bioactive molecules. In this study, we aimed to investigate the therapeutic effects of MSC-exo against HPH and elucidate the underlying molecular mechanism. MethodsExosomes were isolated from conditioned media of human bone mesenchymal stromal cells using ultracentrifugation and characterized through western blotting, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). An HPH animal model was established in male SD rats, and MSC-exo or phosphate-buffered saline (PBS) were administered via the tail vein for three weeks. Subsequently, right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary vascular remodeling were evaluated. Lung tissues from HPH rats and normal rats underwent high-throughput sequencing and transcriptomic analysis. Gene Ontology (GO) analysis was employed to identify upregulated differentially expressed genes. Additionally, rat pulmonary artery smooth muscle cells (PASMC) exposed to platelet-derived growth factor-BB (PDGF-BB) were used to simulate HPH-related pathological behavior. In vitro cellular models were established to examine the molecular mechanism of MSC-exo in HPH. ResultsMSC-exo administration protected rats from hypoxia-induced increases in RVSP, RVHI, and pulmonary vascular remodeling. Additionally, MSC-exo alleviated PDGF-BB-induced proliferation and migration of PASMC. Transcriptomic analysis revealed 267 upregulated genes in lung tissues of HPH rats compared to control rats. Gene Ontology analysis indicated significant differences in pathways associated with Yes Associated Protein 1 (YAP1), a key regulator of cell proliferation and organ size. RT-qPCR and western blot analysis confirmed significantly increased expression of YAP1 in HPH lung tissues and PASMC, which was inhibited by MSC-exo treatment. Furthermore, analysis of datasets demonstrated that Secreted Phosphoprotein 1 (SPP1), also known as Osteopontin (OPN), is a downstream binding protein of YAP1 and can be upregulated by PDGF-BB. MSC-exo treatment reduced the expression of both YAP1 and SPP1. Lentivirus-mediated knockdown of YAP1 inhibited PDGF-BB-induced PASMC proliferation, migration, and SPP1 protein levels. ConclusionOur findings demonstrate that MSC-exo exert a therapeutic effect against hypoxia-induced pulmonary hypertension by modulating the YAP1/SPP1 signaling pathway. The inhibition of YAP1 and downstream SPP1 expression by MSC-exo may contribute to the attenuation of pulmonary vascular remodeling and PASMC proliferation and migration. These results suggest that MSC-exo could serve as a potential therapeutic strategy for the treatment of HPH. Further investigations are warranted to explore the clinical applicability of MSC-exo-based therapies in HPH patients.

Secreted phosphoprotein 1 regulates natural compound 3’,4’,5,7-tetrahydroxyflavone to inhibit mast cell-mediated allergic inflammation

BackgroundMast cells (MCs) are important effector cells in anaphylaxis and anaphylactic disease. 3’,4’,5,7-tetrahydroxyflavone (THF) presents in many medicinal plants and exerts a variety of pharmacological effects. In this study, we evaluated the effect of THF on C48/80-induced anaphylaxis and the mechanisms underlying its effects, including the role of secreted phosphoprotein 1 (SPP1), which has not been reported to IgE-independent MC activation.ResultsTHF inhibited C48/80-induced Ca2+ flow and degranulation via the PLCγ/PKC/IP3 pathway in vitro. RNA-seq showed that THF inhibited the expression of SPP1 and downstream molecules. SPP1 is involved in pseudo-anaphylaxis reactions. Silencing SPP1 affects the phosphorylation of AKT and P38. THF suppressed C48/80-induced paw edema, hypothermia and serum histamine, and chemokines release in vivo.ConclusionsOur results validated SPP1 is involved in IgE-independent MC activation anaphylactoid reactions. THF inhibited C48/80-mediated anaphylactoid reactions both in vivo and in vitro, suppressed calcium mobilization and inhibited SPP1-related pathways.

P-805 Gene expression of human endometrial organoids hormonally treated in vitro for 28 days

Abstract Study question Does gene expression of human endometrial organoids (EO) mimic the in vivo response to a 28- days substitutive hormonal treatment? Summary answer Gene expression profile of human EO exposed to 28-day hormone treatment showed correspondence with the phases of the menstrual cycle in vivo. What is known already The recent development of human EO demonstrated their translational potential for reproductive medicine. Current studies testing functional response to hormones of EO are based on short-term responsiveness to estrogen (E2), progesterone (P4) and differentiation factors up to 10 days. It is unknown how organoids can be used to faithfully reproduce and/or model a complete menstrual cycle of 28 days. High-throughput analyses on endometrium have identified specific markers available for characterizing differentiation and functional aspects related to secretory activity and receptivity to implantation. Study design, size, duration Experimental research was carried out including 4 endometrial biopsies donated by women with normal endometrium function, recruited at an academic center for assisted reproduction between 2022-06-15 to 2022-07-28. These endometrial biopsies were processed for organoid derivation and culture. The EO were hormonally treated in vitro and analyzed for gene expression. Participants/materials, setting, methods Human endometrial biopsies were enzymatically digested to isolate endometrial glands, embedded in matrigel and cultivated in a spinning bioreactor to achieve organoid formation. Thereafter EO were treated as following: 1) E2: (day 0–28); 2) E2 + P4: E2 (day 0–28), P4 (day 14–28); 3) E2 + P4 + dibutyryl cyclic Adenosine monophosphate (dbcAMP): E2 (day 0–28) + P4 (day 14–28) + dbcAMP (day 14–28) or 4) control group. Main results and the role of chance A total of 10 markers were selected for functional analysis and 3 showed a pattern of interest. The target gene expression included Forkhead box O1 (FOXO1), involved in regulating endometrium receptivity in human; progesterone associated endometrium protein (PAEP), a major protein in glandular endometrium secretions; and secreted phosphoprotein 1 (SPP1), an extracellular cytokine that increases in endometrium during the receptive phase. Gene expression of FOXO1, PAEP and SPP1 showed a significant increase in the organoids exposed to complete hormonal cycle, compared to controls. PAEP and SPP1 gene expression was lower up to day 21 but upregulated to 15-fold relative to non-treated group by day 28. This pattern recapitulates a similar response to the physiological conditions in vivo in the human endometrium. Although gene expression patterns followed similar trends, individual biopsies had slight variations in gene expression along 28-day hormone treatment. The 28 day-hormone treatment can stimulate EO for recapitulating histological and functional aspects. The dynamics resembled a timely correlation with gene expression changes accounting for some specific markers mostly involved in receptivity, and thus reinforcing the potential application of EO for further research purposes. Limitations, reasons for caution In vitro organoid culture conditions may have some influence over responsiveness and gene expression that requires further optimization to reproduce uterine environment in vitro and for other conditions related to infertility in a completely defined culture system. Wider implications of the findings Our findings enable the opportunity to expand experimental research over endometrial disorders affecting different stages of the menstrual cycle and also to improve research related to embryo-endometrium interactions. Trial registration number This research has been funded by grants from The Swedish Cancer Society, The Swedish Childhood Cancer Foundation, The Cancer Research Funds of Radiumhemmet, The Swedish Cancer Association BRO, the Stockholm County Council (CIMED) and Karolinska Institutet.

SPP1 and the risk score model to improve the survival prediction of patients with hepatocellular carcinoma based on multiple algorithms and back propagation neural networks

Hepatocellular carcinoma (HCC) is associated with poor prognosis and fluctuations in immune status. Although studies have found that secreted phosphoprotein 1 (SPP1) is involved in HCC progression, its independent prognostic value and immune-mediated role remain unclear. Using The Cancer Genome Atlas and Gene Expression Omnibus data, we found that low expression of SPP1 is significantly associated with improved survival of HCC patients and that SPP1 expression is correlated with clinical characteristics. Univariate and multivariate Cox regression confirmed that SPP1 is an independent prognostic factor of HCC. Subsequently, we found that T cell CD4 memory-activated monocytes, M0 macrophages, and resting mast cells showed significant differences in penetration in the high and low SPP1 expression groups. Next, we used the Weighted Gene Co-Expression Network and Least Absolute Shrinkage Sum Selection Operator algorithms to construct a risk score for the 9-immune-related genes signature. The risk score showed a good ability to identify high and low-risk patients and improved survival prediction. We also used multivariate Cox regression to validate that risk score was significantly correlated with SPP1 and overall survival. Lastly, the Back-Propagation Neural Network confirmed the reliability of the results of multiple algorithms. In conclusion, the findings suggest that SPP1 is an independent marker of HCC survival and immunotherapy.

Immunosuppressive role of SPP1-CD44 in the tumor microenvironment of intrahepatic cholangiocarcinoma assessed by single-cell RNA sequencing.

To demonstrate the biological function of Secreted Phosphoprotein 1(SPP1) and its immune suppressive role in the progression intrahepatic cholangiocarcinoma (ICC). We collected 62,770 cells' published transcriptome data of nine patients whose paired adjacent liver and tumor tissues were both available. We applied differential gene expression analysis to screen potential ICC marker genes, survival analysis to verify the prognostic value of SPP1, and correlation analysis to decipher factors that are related to SPP1 expression. The CellChat was used to distinguish interactions between cancer and T cells. CytoSig was applied to query cytokines that modulate CD44. Further, we established a proliferation score and correlated the score with inhibitory signals to determine the proliferation-suppressive function of SPP1-CD44. SPP1 expression is significantly upregulated in tumoral epitheliums, and patients with higher SPP1 expression have worse survival (P < 0.05). Tumor cells communicate with T cells via SPP1-CD44 interactions. The average expression of SPP1 in malignant cells (SPP1m) and CD44 in T cells (CD44t) is moderately negatively correlated with T cell proliferation score. Immunosuppressive cytokine TGFβ-3 identified as an inducer of CD44 and was significantly negatively correlated with proliferation score (R = -0.88, P < 0.01), and the negative correlation was aggravated in samples with high CD44 expression. SPP1 is a prognostic marker of ICC and is associated with the genome heterogeneity. SPP1-CD44 hinders sustained proliferation of T cells, but immunosuppressive T cells in the tumor microenvironment may evade this inhibition by reducing CD44 expression.

Sacubitril Valsartan Enhances Cardiac Function and Alleviates Myocardial Infarction in Rats through a SUV39H1/SPP1 Axis.

Sacubitril valsartan (lcz696) has been demonstrated as a substitute for angiotensin-converting enzyme inhibitors and angiotensin receptor blockers for the treatment of heart failure. This research is aimed at examining the effects of lcz696 and its target molecules on myocardial infarction (MI). A rat model of MI was induced by left anterior descending artery ligation and treated with lcz696. Lcz696 treatment significantly reduced cardiac injury and heart failure, restored the left ventricular fractional shortening and ejection fraction, and reduced oxidative stress and inflammatory responses in rat myocardium. By analyzing the heart failure-related GSE47495 dataset and performing gene ontology (GO) functional enrichment analysis, we obtained histone lysine methyltransferase SUV39H1 and secreted phosphoprotein 1 (SPP1) as two molecules implicated in the oxidative stress and inflammation processes. An elevation of SUV39H1 whereas a decline of SPP1 were detected in cardiac tissues after lcz696 treatment. Enrichments of SUV39H1 and H3K9me3 at the SPP1 promoter were identified by chromatin immunoprecipitation assay. SUV39H1 catalyzed H3K9me3 modification to suppress the expression of SPP1. Preconditioning of SUV39H1 silencing blocked the protective roles of lcz696, but SPP1 silencing alleviated the myocardial injury. In conclusion, this study demonstrates that lcz696 enhances cardiac function and alleviates MI in rats through a SUV39H1/SPP1 axis.

Overexpression of Secreted Phosphoprotein 1 (SPP1) predicts poor survival in HPV positive cervical cancer

Cervical cancer (CC) is the most prevalent malignant gynecological tumor with limited treatments. The present study describes the role of SPP1 in cancer progression, SPP1 emerged as one of the most overexpressed genes identified through clariom D transcriptome microarray. This investigation aims towards identifying a potential gene with significant prognostic value for detection and early diagnosis of cervical cancer. The elevated expression of SPP1 in cervical squamous cell carcinoma tissue was validated across GEO (Gene Expression Omnibus) microarray data sets, TCGA (The Cancer Genome Atlas), and Oncomine databases. SPP1 expression was found to be prognostically significant, showing association with poor survival rate of the patients. Our study intended to assess the expression of secreted phosphoprotein (SPP1) gene at mRNA and protein levels, and to explore the association of single nucleotide polymorphisms of SPP1 with risk of CC. Further, receiver operating characteristics (ROC) curve was plotted to determine the levels of SPP1 to differentiate CC against control. Results revealed significant (p < 0.01) stage-wise upregulation of SPP1 in CC compared to the normal cervical tissue and this was further confirmed using Immunohistochemistry and real-time PCR. The ROC for SPP1 demonstrated good selective power to differentiate malignant CC and non-malignant cervical tissues. The SPP1 gene −443 T > C promoter polymorphisms are found to be significantly predominant in the disease group and Insilico analysis by the TRANSFAC software confirms its association with loss of STAT6 transcription factor binding site leading to overexpression of the SPP1.

Long-term running exercise improves cognitive function and promotes microglial glucose metabolism and morphological plasticity in the hippocampus of APP/PS1 mice

BackgroundThe role of physical exercise in the prevention of Alzheimer’s disease (AD) has been widely studied. Microglia play an important role in AD. Triggering receptor expressed in myeloid cells 2 (TREM2) is expressed on microglia and is known to mediate microglial metabolic activity and brain glucose metabolism. However, the relationship between brain glucose metabolism and microglial metabolic activity during running exercise in APP/PS1 mice remains unclear.MethodsTen-month-old male APP/PS1 mice and wild-type mice were randomly divided into sedentary groups or running groups (AD_Sed, WT_Sed, AD_Run and WT_Run, n = 20/group). Running mice had free access to a running wheel for 3 months. Behavioral tests, [18]F-FDG-PET and hippocampal RNA-Seq were performed. The expression levels of microglial glucose transporter (GLUT5), TREM2, soluble TREM2 (sTREM2), TYRO protein tyrosine kinase binding protein (TYROBP), secreted phosphoprotein 1 (SPP1), and phosphorylated spleen tyrosine kinase (p-SYK) were estimated by western blot or ELISA. Immunohistochemistry, stereological methods and immunofluorescence were used to investigate the morphology, proliferation and activity of microglia.ResultsLong-term voluntary running significantly improved cognitive function in APP/PS1 mice. Although there were few differentially expressed genes (DEGs), gene set enrichment analysis (GSEA) showed enriched glycometabolic pathways in APP/PS1 running mice. Running exercise increased FDG uptake in the hippocampus of APP/PS1 mice, as well as the protein expression of GLUT5, TREM2, SPP1 and p-SYK. The level of sTREM2 decreased in the plasma of APP/PS1 running mice. The number of microglia, the length and endpoints of microglial processes, and the ratio of GLUT5+/IBA1+ microglia were increased in the dentate gyrus (DG) of APP/PS1 running mice. Running exercise did not alter the number of 5-bromo-2′-deoxyuridine (BrdU)+/IBA1+ microglia but reduced the immunoactivity of CD68 in the hippocampus of APP/PS1 mice.ConclusionsRunning exercise inhibited TREM2 shedding and maintained TREM2 protein levels, which were accompanied by the promotion of brain glucose metabolism, microglial glucose metabolism and morphological plasticity in the hippocampus of AD mice. Microglia might be a structural target responsible for the benefits of running exercise in AD. Promoting microglial glucose metabolism and morphological plasticity modulated by TREM2 might be a novel strategy for AD treatment.

Secreted phosphoprotein 1 as a potential prognostic and immunotherapy biomarker in multiple human cancers

ABSTRACT Secreted phosphoprotein 1 (SPP1) is involved in immune regulation, cell survival, and tumor progression. Studies have demonstrated that SPP1 plays an important role in certain individual tumors. However, the expression profile and oncogenic features of SPP1 in diverse cancers are remaining unknown. Therefore, we performed a comprehensive analysis using The Cancer Genome Atlas (TCGA) database. Raw data of 33 cancer types were download from the University of California Santa Cruz (UCSC) Xena website. The expression of SPP1 and its relationship with tumor prognosis, immune invasion, tumor microenvironment, and immunotherapy were analyzed using the R language. The function analysis was conducted using Gene Set Enrichment Analysis (GSEA). The oncogenic features of SPP1 was validated by wound-healing assay and EdU staining assay. SPP1 highly expressed in most cancers. The expression of SPP1 was significant related to prognosis, tumor mutation burden (TMB), microsatellite instability (MSI), and immune checkpoint genes, suggested that SPP1 plays an essential role in the tumor immune microenvironment and immune cell infiltration. The immune/stromal scores correlated positively with the SPP1 expression, and the relationship was affected by tumor heterogeneity and immunotherapy. In addition, SPP1 could predict the response of tumor immunotherapy. Functional analysis revealed the SPP1-associated terms and pathways. Finally, SPP1 significantly elevated cell proliferation and migration in A549, Huh7, HT-29, A2780 tumor cell lines. In conclusion, this study indicated that SPP1 involved in tumorigenesis, tumor progression, and regulated tumor immune microenvironment, revealing SPP1 might be a potential target for evaluating prognosis and immunotherapy in multiple cancers.

LncRNA-XIST Promotes Proliferation and Migration in ox-LDL Stimulated Vascular Smooth Muscle Cells through miR-539-5p/SPP1 Axis.

Long noncoding RNAs (lncRNAs) are untranslated transcripts greater than 200 nucleotides in length. Despite not being translated, they play a role in the regulation of transcription, translation, and other cellular processes and have been identified as key regulator in the progression of atherosclerosis. This study focused on the lncRNA X-inactive specific transcript (XIST), which participates in the regulation of X chromosome inactivation. XIST is produced by the XIST gene and is located on human chromosome Xql3.2. We also focused on discovering the possible role and mechanism of lncRNA XIST in oxidized low-density lipoprotein- (ox-LDL-) stimulated vascular smooth muscle cells (VSMCs), which could further help evalute its possible a role in the progression of atherosclerosis. XIST was overexpressed in ox-LDL-stimulated VSMCs, while the expression of miR-539-5p was decreased. XIST knockdown hindered the proliferation and migration of ox-LDL-treated VSMCs. XIST inhibits the miR-539-5p expression through direct interaction. Besides, miR-539-5p inhibitors can partially reverse the effect of XIST depletion on the proliferation and migration of VSMCs induced by ox-LDL stimulation. Further mechanistic analysis showed that secreted phosphoprotein 1 (SPP1) is the target of miR-539-5p, and XIST acts as a competing endogenous RNA for miR-539-5p to enhance the expression of SPP1. In addition, miR-539-5p inhibitor exerts its proliferation and migration effects by activating the miR-539-5p/SPP1 axis in VSMCs stimulated by ox-LDL. In conclusion, our study findings show that XIST inhibition can inhibit the proliferation and migration of atherosclerosis vascular smooth muscle cells, which provides a new theoretical basis for atherosclerosis treatment.

Higher Expression of SPP1 Predicts Poorer Survival Outcomes in Head and Neck Cancer.

Secreted phosphoprotein 1 (SPP1) participated in various biological processes in many cancers, including immune response, tumor progression, and prognosis. However, SPP1 in head and neck squamous cell carcinoma (HNSCC) remains unknown. Clinical-genetic data of HNSCC were obtained from The Cancer Genome Atlas (TCGA) database. The differential expression of SPP1 in HNSCC tissues and adjacent normal tissues was quantified by bioinformatics methods and verified by western blot and other differential biological methods. We concluded that SPP1 is significantly upregulated in tumor tissues and can become a prognostic biomarker for HNSCC.

The Correlation Between SPP1 and Immune Escape of EGFR Mutant Lung Adenocarcinoma Was Explored by Bioinformatics Analysis.

BackgroundImmune checkpoint inhibitors have achieved breakthrough efficacy in treating lung adenocarcinoma (LUAD) with wild-type epidermal growth factor receptor (EGFR), leading to the revision of the treatment guidelines. However, most patients with EGFR mutation are resistant to immunotherapy. It is particularly important to study the differences in tumor microenvironment (TME) between patients with and without EGFR mutation. However, relevant research has not been reported. Our previous study showed that secreted phosphoprotein 1 (SPP1) promotes macrophage M2 polarization and PD-L1 expression in LUAD, which may influence response to immunotherapy. Here, we assessed the role of SPP1 in different populations and its effects on the TME.MethodsWe compared the expression of SPP1 in LUAD tumor and normal tissues, and in samples with wild-type and mutant EGFR. We also evaluated the influence of SPP1 on survival. The LUAD data sets were downloaded from TCGA and CPTAC databases. Clinicopathologic characteristics associated with overall survival in TCGA were assessed using Cox regression analysis. GSEA revealed that several fundamental signaling pathways were enriched in the high SPP1 expression group. We applied CIBERSORT and xCell to calculate the proportion and abundance of tumor-infiltrating immune cells (TICs) in LUAD, and compared the differences in patients with high or low SPP1 expression and wild-type or mutant EGFR. In addition, we explored the correlation between SPP1 and CD276 for different groups.ResultsSPP1 expression was higher in LUAD tumor tissues and in people with EGFR mutation. High SPP1 expression was associated with poor prognosis. Univariate and multivariate cox analysis revealed that up-regulated SPP1 expression was independent indicator of poor prognosis. GSEA showed that the SPP1 high expression group was mainly enriched in immunosuppressed pathways. In the SPP1 high expression group, the infiltration of CD8+ T cells was lower and M2-type macrophages was higher. These results were also observed in patients with EGFR mutation. Furthermore, we found that the SPP1 expression was positively correlated with CD276, especially in patients with EGFR mutation.ConclusionSPP1 levels might be a useful marker of immunosuppression in patients with EGFR mutation, and could offer insight for therapeutics.

Upregulation of SPP1 Is a Marker for Poor Lung Cancer Prognosis and Contributes to Cancer Progression and Cisplatin Resistance.

The chemoresistance of lung cancer is a significant contributor to its high mortality and morbidity rate. There is an urgent need to identify differentially expressed genes in lung cancer patients with a poor prognosis to develop effective means to overcome drug resistance in subsequent treatment. In this study, we identified the secreted phosphoprotein 1 (SPP1) as a potential gene associated with a poor diagnosis of lung cancer patients using the Cancer Genome Atlas analysis, which suggested that the expression of SPP1 in tumor tissues was significantly higher than normal tissues. The high expression of SPP1 was also correlated with tumor grade and poor clinical prognosis. To understand the roles of SPP1 and the DNA methyltransferase 1 (DNMT1), which regulated SPP1 expression, in affecting cell viability, migration and invasion, SPP1 and DNMT1 were overexpressed in the human lung cancer A549 and NCI-446 cells, followed by analyzing cell viability, migration and invasion. We showed that SPP1 promoted the proliferation, migration and invasion of lung cancer cells, and increased the resistance of lung cancer to the chemotherapeutic drug cisplatin. Knocking down SPP1 in cells restored sensitivity to cisplatin. Further, A549 cells without SPP1 overexpression demonstrated lower tumor growth rate than SPP1 overexpression cells using the xenograft tumor mouse model. High expression of SPP1 in lung cancer tumor tissue was caused by the reduced methylation level of its promoter region mediated by DNMT1. Our data suggested that SPP1 can be used as a marker for highly malignant lung cancer and targeting SPP1 may be a potential lung cancer treatment strategy.

Loss of ITGB3 in ovine conceptuses decreases conceptus expression of NOS3 and SPP1: implications for the developing placental vasculature†.

During the peri-implantation period of pregnancy in sheep, there is an initial period of loose apposition of the elongating conceptuses (embryos and associated placental membranes) to the endometrial luminal epithelium (LE) that is followed by adhesion of the conceptus trophectoderm to the endometrial LE for implantation. Integrins and maternal extracellular matrix (ECM) molecules are major contributors to stable adhesion at implantation, and the β3 integrin subunit (ITGB3) is implicated in the adhesion cascade for implantation in several species including the sheep. We blocked mRNA translation for trophectoderm-expressed ITGB3 by infusing morpholino antisense oligonucleotides into the uterine lumen of pregnant ewes on Day 9 to assess effects on conceptus elongation, and on Day 16 to assess effects on early placental development in sheep. Results indicate that sheep conceptuses elongate and implant to the uterine wall in the absence of ITGB3 expression by the conceptuses; however, loss of ITGB3 in conceptuses decreased the growth of embryos to Day 24 of gestation, and decreased expression of secreted phosphoprotein 1 (SPP1) and nitric oxide synthase 3 (NOS3). Abundant SPP1 was localized around the blood vessels in the placental allantoic membrane in normal sheep pregnancies. We hypothesize that NOS3 and SPP1 positively influence the development of the vasculature within the allantois, and that decreased expression of NOS3 and SPP1, in response to knockdown of ITGB3 in conceptuses, alters development of the vasculature in the allantois required to transport nutrients from the endometrium to support growth and development of the embryo.

Analysis of Expression and Its Clinical Significance of the Secreted Phosphoprotein 1 in Lung Adenocarcinoma.

ObjectiveTo explore the expression of secreted phosphoprotein 1 (SPP1) in lung adenocarcinoma (LUAD), and evaluate its relationship with clinicopathological characteristics and prognosis of LUAD, and analyze the advantages of SPP1 as a potential prognostic marker in LUAD.MethodsThe expression of SPP1 in normal lung tissue and LUAD was analyzed from the Cancer Cell Line Encyclopedia (CCLE), Gene Expression Omnibus (GEO), and Human Protein Atlas (HPA) databases. GSE68465 was used to explore the relationship between the SPP1 expression and clinicopathological characteristics and the prognosis of LUAD patients. The relationship between SPP1 and immune infiltration in LUAD was analyzed by the Tumor Immune Estimation Resource (TIMER) database. Gene enrichment analysis was performed in GSEA. The Cancer Genome Atlas (TCGA)-LUAD data was used to verify the results.ResultsIn the cell line level, non-small cell lung cancer ranked ninth among cancer cell lines based on SPP1 expression. In the messenger RNA (mRNA) and protein levels, SPP1 expression was higher in LUAD tissues than that in normal control. SPP1 expression was related to gender, N stage, histological grade, and progression or relapse. In men, SPP1 expression were higher compared to that in women. The higher the N stage, the higher the SPP1 expression level. As LUAD progresses or relapses, SPP1 expression could increase. In the pathological grade, the SPP1 expression was higher in LUAD samples with moderate differentiation. In addition, the overall 5-year survival rates of the SPP1 high and low expression groups were 50.574 and 59.181% [P = 0.008; hazard ratio (HR) = 0.7057; 95% CI, 0.5467–0.9109], indicating that SPP1 had an impact on overall survival for LUAD patients. The relationship between SPP1 expression and CD4+ T cell, macrophage, neutrophil, and dendritic cell infiltration was weak in LUAD. SPP1 could be considered as an independent prognostic marker in LUAD (P = 0.003; HR = 1.150; 95% CI, 1.048–1.261) by multivariate Cox regression analysis. The results of GSEA indicated that samples with high SPP1 expression were enriched in protein secretion, mTORC1 signaling, angiogenesis, and glycolysis pathway. The analysis results obtained by TCGA-LUAD data were basically consistent with the results obtained by GSE68465.ConclusionsSPP1 can not only affect the occurrence and development of LUAD but also may be an independent prognostic marker of LUAD. SPP1 is expected to be a new target for molecular targeted therapy.

The underlying mechanisms of lorlatinib penetration across the blood-brain barrier and the distribution characteristics of lorlatinib in the brain.

ObjectiveTo clarify the distribution of lorlatinib in the brain and elucidate the molecular mechanisms of lorlatinib penetration across the blood‐brain barrier (BBB).MethodsCytological experiments were performed to investigate the growth inhibitory effect of lorlatinib on different cells (endothelial cells HUVEC, HMEC‐1, and HCMEC/D3) and to investigate the protective effect of lorlatinib on neuronal cells after SH‐SY5Y hypoxia/reoxygenation injury. Furthermore, rat brain tissue was sequenced, and the differentially expressed genes (secreted phosphoprotein 1 (SPP1), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF‐β), Claudin, ZO‐1 and P‐gp) in several different drug treatment groups were verified by Real‐Time PCR. Lorlatinib brain distribution was predicted by physiologically based pharmacokinetics (PBPK).ResultsLorlatinib and crizotinib both had inhibitory effects on endothelial cells, however lorlatinib inhibited the growth of HCMEC/D3 more efficaciously than crizotinib. In the SH‐SY5Y hypoxia model, lorlatinib had a greater protective effect on nerve cell damage caused by hypoxia and reoxygenation than crizotinib. The expression of SPP1, VEGF, TGF‐β, and Claudin in brain tissue was significantly downregulated after lorlatinib administration, and the expression level of early growth transcription factor 1 (Egr‐1) was significantly increased. The PBPK model successfully described lorlatinib concentrations in blood and brain tissue in the mouse model and gave a brain tissue partition coefficient of 0.7.ConclusionLorlatinib can increase the permeability of the blood‐brain barrier whereby we suggest its underlying working mechanism is related to downregulating SPP1, inhibiting VEGF, TGF‐β, and Claudin subsequently reducing the number of tight junctions between BBB cells. Lorlatinib plays a protective role on injured nerve cells and does not change the amount of P‐gp expression in brain tissue, which may be important for its ability to be efficacious across the BBB with a low incidence of resistance.

Follistatin-like Protein 1 Inhibits Lung Cancer Metastasis by Preventing Proteolytic Activation of Osteopontin.

Follistatin-like protein 1 (FSTL1) plays a critical role in lung organogenesis, but is downregulated during lung cancer development and progression. The prognostic significance and functional consequences of FSTL1 downregulation in lung cancer are unclear. Here, reduced levels of FSTL1 were detected in various tumors compared with normal tissues and were associated with poor clinical outcome in patients with non-small cell lung cancer, particularly those with lung adenocarcinoma. FSTL1 expression negatively correlated with the metastatic potential of lung cancer cells. Antibody-based neutralization of extracellular FSTL1 increased cellular migration/invasion while addition of recombinant FSTL1 protein diminished the metastatic capacity of lung cancer cells in vitro and in vivo. Notably, treatment with FSTL1 effectively prevented the metastatic progression of lung cancer cells in an orthotopic animal model. Mechanistically, FSTL1 directly bound to the proform of secreted phosphoprotein 1 (SPP1)/osteopontin, restraining proteolytic activation of SPP1, which led to inactivation of integrin/CD44-associated signaling and rearrangement of the actin cytoskeleton. Combined low expression of FSTL1 and high expression of SPP1 predicted a poorer prognosis for patients with lung cancer. This study highlights the novel interaction between FSTL1 and SPP1 and new opportunities to effectively target SPP1-driven metastatic cancers characterized by FSTL1 downregulation. SIGNIFICANCE: These findings describe the novel interaction between FSTL1 and SPP1 and its role in the metastatic progression of lung adenocarcinoma.

Identification of Secreted Phosphoprotein 1 (SPP1) as a Prognostic Factor in Lower-Grade Gliomas

Secreted phosphoprotein 1 (SPP1) is an important extracellular glycoprotein that is associated with immune regulation, tumorigenesis, and cell signaling. However, the prognostic value of SPP1 in patients with glioma has not yet been clarified, especially in lower-grade gliomas. The objective of this study is to evaluate the prognostic merit of SPP1 in lower-grade gliomas. The messenger RNA (mRNA) expression of SPP1 in about 1000 cancer cell lines was explored by using the data from the Cancer Cell Line Encyclopedia database. The Oncomine database was mined to evaluate the mRNA expression of SPP1 in lower-grade glioma, glioblastoma, and normal brain tissues. The correlation between SPP1 mRNA expression and overall survival of patients with glioma from The Cancer Genome Atlas database was analyzed. SPP1 mRNA expression of glioma was ranked as the eighth highest of all cancer cell lines in the Cancer Cell Line Encyclopedia database. The data from the Oncomine database suggested that SPP1 expression was significantly high in glioblastoma compared with normal brain tissues but was not significantly high in lower-grade glioma compared with normal brain tissue. Analysis of the RNA-Seq data from The Cancer Genome Atlas database showed that the increased SPP1 mRNA expression in lower-grade glioma was significantly associated with poor survival outcomes in patients with lower-grade glioma. Multivariate Cox regression analysis showed that SPP1 might be considered as an independent prognostic factor in lower-grade gliomas. The present study showed that SPP1 overexpression is related to worse overall survival in patients with lower-grade glioma. Moreover, SPP1 could be considered as an independent factor in lower-grade gliomas.