Secreted Protein Acidic And Rich In Cysteines-like 1 Research Articles

Serum Secreted Protein Acidic and Rich in Cysteine-Like 1 as a Biochemical Predictor for Prognosticating Clinical Outcomes After Acute Supratentorial Intracerebral Hemorrhage: A Prospective Cohort Study.

Secreted protein acidic and rich in cysteine-like 1 (SPARCL1) regulates synaptic stability and is up-regulated during axonal regeneration. Here, serum SPARCL1 was determined for estimating severity and prognosticating early neurological deterioration (END) and functional outcomes of acute intracerebral hemorrhage (ICH). In this prospective observational cohort study of 156 patients with supratentorial ICH, blood samples of 53 were acquired not only at admission but also ad days 1, 3, 5, 7 and 10. Another group of 53 healthy controls were recruited. The modified Rankin Scale (mRS) scores of 3-6 at poststroke six months were regarded as poor prognosis. As opposed to controls, serum SPARCL1 levels were markedly elevated during the early ten days after ICH, with the highest levels at days 1 and 3. Admission serum SPARCL1 levels were independently correlated with National Institutes of Health Stroke Scale scores and hematoma volume, were significantly increased in the order of six-month mRS scores from 0 to 6 and were independently correlated with six-month mRS scores. Serum SPARCL1 levels were linearly related to risks of poor six-month prognosis and END under restricted cubic spline, had significant efficiency under receiver operating characteristic (ROC) curve and were independently associated with END and poor prognosis. Subgroup analysis confirmed that no interactions existed for associations of serum SPARCL1 levels with other variables, such as age, gender and some specific vascular risk factors. END and poor prognosis prediction models integrating serum SPARCL1 were displayed using the two nomograms. The poor prognosis prediction model, but END prediction model not, performed well under calibration curve, decision curve and ROC curve. A substantial elevation of serum SPARCL1 levels during the early period after ICH is independently related to illness severity and poor neurological outcomes, thus signifying that serum SPARCL1 may appear as a prognostic biomarker of ICH.

Identifying liver metastasis-related hub genes in breast cancer and characterizing SPARCL1 as a potential prognostic biomarker.

The liver is the third most common metastatic site for advanced breast cancer (BC), and liver metastases predict poor prognoses. However, the characteristic biomarkers of BC liver metastases and the biological role of secreted protein acidic and rich in cysteine-like 1 (SPARCL1) in BC remain unclear. The present study aimed to identify potential biomarkers for liver metastasis of BC and to investigate the effect of SPARCL1 on BC. The publicly available GSE124648 dataset was used to identify differentially expressed genes (DEGs) between BC and liver metastases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to annotate these DEGs and understand the biological functions in which they are involved. A protein-protein interaction (PPI) network was constructed to identify metastasis-related hub genes and further validated in a second independent dataset (GSE58708). Clinicopathological correlation of hub gene expression in patients with BC was determined. Gene set enrichment analysis (GSEA) was performed to explore DEG-related signaling pathways. SPARCL1 expression in BC tissues and cell lines was verified by RT-qPCR. Further in vitro experiments were performed to investigate the biological functions of SPARCL1 in BC cells. We identified 332 liver metastasis-related DEGs from GSE124648 and 30 hub genes, including SPARCL1, from the PPI network. GO and KEGG enrichment analyses of liver-metastasis-related DEGs revealed several enriched terms associated with the extracellular matrix and pathways in cancer. Clinicopathological correlation analysis of SPARCL1 revealed that its expression in BC was associated with age, TNM stage, estrogen receptor status, progesterone receptor status, histological type, molecular type, and living status of patients. GSEA results suggested that low SPARCL1 expression in BC was related to the cell cycle, DNA replication, oxidative phosphorylation, and homologous recombination. Lower expression levels of SPARCL1 were detected in BC tissues compared to adjacent tissues. The in vitro experiments showed that SPARCL1 knockdown significantly increased the proliferation and migration of BC cells, whereas the proliferation and migration were suppressed after elevating the expression of SPARCL1. We identified SPARCL1 as a tumor suppressor in BC, which shows potential as a target for BC and liver metastasis therapy and diagnosis.

Immunological role and prognostic value of SPARCL1 in pan-cancer analysis.

Background: Secreted protein acidic and rich in cysteine-like 1 (SPARCL1) was a kind of extracellular matrix glycoprotein. SPARCL1 was strongly inhibited in most cancers. However, the potential functions of SPARCL1 in the pan-cancer cohort have not been widely studied. Methods: We evaluated the transcriptional level and the prognostic value of SPARCL1 in 33 types of cancer and revealed the relationship between genetic alterations of SPARCL1 and the tumor mutation burden. Meanwhile, we assessed the correlations between SPARCL1 and tumor-infiltrating lymphocytes across cancers. Results: The transcriptional level of SPARCL1 was inhibited in most cancers. Although SPARCL1 was down-regulated in most cancers, SPARCL1 might play a protective or detrimental role in different cancers. We demonstrated that mutation count was elevated in the altered SPARCL1 group in several cancers. Additionally, we found a significant positive correlation between SPARCL1 and macrophage infiltration levels in most cancers. Especially, marker sets of M2 macrophages were strongly related to SPARCL1 in cholangiocarcinoma, colon adenocarcinoma, rectum adenocarcinoma, and pancreatic adenocarcinoma. Conclusion: Our study found that SPARCL1 might work as a biomarker for prognosis and immune infiltration in pan-cancer analysis.

SPARCL1 Is a Novel Prognostic Biomarker and Correlates with Tumor Microenvironment in Colorectal Cancer

Background Secreted protein acidic and rich in cysteine-like 1 (SPARCL1) plays an important role in tumor pathogenesis. We aim to evaluate the clinical significance and potential biological roles of SPARCL1 in colorectal cancer (CRC). Methods Datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were downloaded to evaluate the expression levels of SPARCL1 in CRC. Receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic value of SPARCL1. Then, comprehensive database search was conducted for published clinical studies to explore clinical significance of SPARCL1. In addition, coexpression genes of SPARCL1 were identified through the cBioPortal database and enrichment analysis of SPARCL1 and its coexpression genes were performed by the “clusterProfiler” R package. Finally, the correlations between SPARCL1 and tumor microenvironment scores, tumor-infiltrating immune cells in CRC were determined by “ESTIMATE” and “GSVA” R packages. Results SPARCL1 was significantly downregulated in CRC tissues, and SPARCL1 showed high accuracy for diagnosis of primary CRC in both GEO and TCGA datasets. Pooled results from published clinical studies showed SPARCL1 expression was associated with differentiation (OR = 1.89, 95% CI: 1.38-2.59), tumor stage (OR = 0.47, 95% CI: 0.29-0.77), distant metastasis (OR = 0.53, 95% CI: 0.33-0.84), and overall survival (HR = 0.56, 95% CI: 0.43-0.74). SPARCL1 and its top 300 coexpression genes were involved in several KEGG pathways, such as focal adhesion, cell adhesion molecules, PI3K-Akt signaling pathway, cGMP-PKG signaling pathway, and ECM-receptor interaction. Besides, the SPARCL1 expression was significantly correlated with stromal score, immune score, ESTIMATE score, and diverse immune cells. Conclusion SPARCL1 significantly correlated with clinicopathological features and tumor microenvironment in CRC.

The inhibition of HeLa cells proliferation through SPARCL1 mediated by SPP1.

Secreted protein acidic and rich in cysteines-like 1 (SPARCL1) is implicated in tumor progression and considered as a tumor suppressor. Aim of the study is to investigate the role of SPARCL1 in the regulation of tumor biology. SPARCL1 expression in human cervical cells was determined through western blot and RT-PCR. The effects of SPARCL1 overexpression on cell proliferation, migration and invasion were evaluated through CCK8 assay, colony formation assay, Wound healing assay and Transwell assay, respectively. The gain function of Secreted phosphor protein 1 (SPP1) was also evaluated in these cell functions. We observed that SPARCL1 expression at protein levels and transcription levels was lower in HeLa cells than that in Ect1/E6E7 cells. When SPARCL1 was overexpressed in HeLa cells, cell proliferation, migration and invasion were greatly repressed. Additionally, SPARCL1 overexpression markedly downregulated SPP1 expression at transcription levels. Mechanistical study revealed that SPP1 overexpression could greatly counteract the effects of SPARCL1 overexpression on the aforementioned cell processes and inhibit the phosphorylation of focal adhesion kinase (FAK) and extracellular regulated protein kinases (ERK). Our findings indicated that HeLa cells overexpressing SPARCL1 showed weaker abilities of proliferation, migration and invasion, and its effects could be neutralized by SPP1 overexpression possibly via FAK/ERK pathway. The relationship of SPARCL1 and SPP1 could help us to further understand the pathogenesis of cervical cancer and SPARCL1/SPP1 could be beneficial therapeutic targets in cervical cancer.

Effects of SPARCL1 on the proliferation and differentiation of sheep preadipocytes

ABSTRACT Important candidate genes that regulate lipid metabolism have the potential to increase the content of intramuscular fat (IMF) and improve meat quality. Secreted protein acidic and rich in cysteine like 1(SPARCL1) is a secreted glycoprotein with important physiological functions and is involved in the proliferation and differentiation of various cells. However, the role of the SPARCL1 gene in sheep preadipocytes and its regulatory mechanism is still unclear. In this study, we explored the effect of SPARCL1 on the proliferation and differentiation of sheep preadipocytes. The results showed that the expression level of the SPARCL1 gene is higher in fat tissue than in other tissues, and the gene was significantly increased on the 6th day of preadipocyte differentiation. In the preadipocyte proliferation stage, interference of SPARCL1 gene reduced cell viability and increased cell apoptosis. In preadipocyte differentiation stage, SPARCL1 overexpression significantly inhibited lipid droplets accumulation and triglyceride content by increasing Wnt10b, Fzd8, IL6, and β-catenin and inhibiting PPARγ, C/EBPα, LPL, and IGF1 genes expression, whereas SPARCL1 deficiency significantly promoted cell differentiation by inhibiting β-catenin and increasing GSK3β, PPARγ, C/EBPα, and LPL. The results of this study suggest that SPARCL1 plays a negative role during preadipocyte differentiation and may become a novel target for regulating preadipocyte differentiation and improving IMF. Abbreviations: IMF: Intramuscular fat SPARCL1 : Secreted protein acidic and rich in cysteine like 1 PPARγ : Peroxisome proliferator-activated receptor γ C/EBPα : CCAAT/enhancer-binding protein-α LPL : Lipoprotein lipase IGF1 : Insulin-like growth factor 1 Wnt10b : Wnt family member 10B Fzd8 : Frizzled class receptor 8 IL6: Interleukin 6 β-catenin : Catenin beta interacting protein 1 GSK3β : Glycogen synthase kinase 3 beta LRP5/6 : Low-density lipoprotein receptor-related protein 5/6

SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway.

As an extracellular matrix protein, secreted protein acidic and rich in cysteine (SPARC)-like 1 (SPARCL1) is involved in various cell functions. It was previously implicated in bovine skeletal muscle-derived satellite cell (MDSC) differentiation; however, the underlying mechanism remains unknown. In this study, immunoprecipitation and mass spectrometry revealed that integrin β1 (ITGB1) combines with SPARCL1. Further, co-immunoprecipitation demonstrated that SPARCL1 interacts with ITGB1. Cell scratch assays explored the influence of SPARCL1 on MDSC migration through ITGB1. In addition, desmin staining for myotube fusion rate and MyoD protein expression results showed that SPARCL1 promotes MDSC early differentiation through ITGB1. Furthermore, Western blotting results demonstrated that SPARCL1 regulates the expression of p-FAK, p-paxillin, vinculin, Cdc42, and Arp2/3 through ITGB1. These findings indicate that SPARCL1 may influence bovine MDSC migration and differentiation through an ITGB1-mediated cell signaling pathway. Herein, we elucidated the mechanism through which SPARCL1 affects MDSC differentiation. Our results provide insight into the molecular mechanism of muscle development and may in the future facilitate skeletal muscle regeneration and treatment.

SPARCL1 impedes trophoblast migration and invasion by down-regulating ERK phosphorylation and AP-1 production and altering EMT-related molecule expression

IntroductionEmbryo implantation depends on trophoblast cells migration and invasion. Abnormal function of trophoblast cells could result in many pregnancy complications. Secreted protein acidic and rich in cysteine like-1 (SPARCL1) has been reported to inhibit cell migration and tumor invasion. This study aimed to explore the role of SPARCL1 in trophoblast functions. MethodsVillous specimens were obtained from 31 women with spontaneous abortion and 31 women with normal early pregnancy to determine the expression of SPARCL1. HTR8/SVneo cells and JAR cells were transfected with pIRES2-EGFP-SPARCL1 vectors and control vectors. The proliferation assay and scratch-wound assay were performed. Quantitative polymerase chain reaction (qPCR) and western blotting were performed to assess epithelial mesenchymal transition (EMT)-related molecules including MMP2, MMP3, N-cadherin, E-cadherin and vimentin. Extracellular signal-regulated kinase (ERK) phosphorylation activity and AP-1 expression in HTR8/SVneo cells following multi-scratching were detected using above assays. ResultsThe mRNA and protein levels of SPARCL1 were significantly higher in the abortion group than in the normal pregnancy group. After transfection, there was no difference of cell viability between the SPARCL1-overexpression group and control vector group. However, the migration distance and area were reduced and the abundances of EMT related molecules were changed by SPARCL1 overexpression when compared with controls. Lower ERK phosphorylation activity and decreased Fos and Jun expressions were noted at high level of SPARCL1. ConclusionRestrained migration and invasion were noted in trophoblast cells with SPARCL1 overexpression, which might affect embryo implantation and placenta development. It could be involved in the pathogenesis of spontaneous abortion.

Pre-analytical stability of novel cerebrospinal fluid biomarkers

Stability of the cerebrospinal fluid (CSF) composition under different pre-analytical conditions is relevant for the diagnostic potential of biomarkers. Our aim was to examine the pre-analytical stability of promising CSF biomarkers that are currently evaluated for their discriminative use in various neurological diseases.Pooled CSF was aliquoted and experimentally exposed to delayed storage: 0, 1, 2, 4, 24, 72, or 168 h at 4 °C or room temperature (RT), or 1–4 months at −20 °C; or up to 7 freeze/thaw (f/t) cycles, before final storage at −80 °C. Eleven CSF biomarkers were screened using immunoassays, liquid chromatography, or enzymatic methods.Levels of neurogranin (truncP75), chitinase-3-like protein (YKL-40), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), acetylcholinesterase (AChE) enzymatic activity, theobromine, secreted protein acidic and rich in cysteine-like 1 (SPARCL-1) and homovanillic acid (HVA) levels were not affected by the applied storage conditions. 3-Methoxy-4-hydroxyphenylglycol (MHPG) levels linearly and strongly decreased after 4 h at RT (−10%) or 24 h at 4 °C (−27%), and with 6% after every f/t cycle. 5-Methyltetrahydrofolate (5-MTHF) (−29% after 1 week at RT) and 5-hydroxyindoleacetic acid levels (5-HIAA) (−16% after 1 week at RT) were reduced and 3,4-dihydroxyphenylacetic acid (DOPAC) levels (+22% after 1 week at RT) increased, but only after >24 h at RT.Ten out of eleven potential CSF novel biomarkers showed very limited change under common storage and f/t conditions, suggesting that these CSF biomarkers can be trustfully tested under the pre-analytical conditions present across different cohorts.

Abstract 195: SPARCL1 is an angiocrine inhibitor of tumorigenesis in colorectal carcinoma

Abstract Colorectal carcinoma (CRC) is the second leading cause of cancer-related mortality worldwide. The contribution of the tumor microenvironment (TME) to CRC pathogenesis is well established, whereby the dominance of one immune response (Th1) over another (Th17) can yield opposite effects on patient prognosis. We recently reported on TME-dependent vascular plasticity in CRC and identified the secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) as a marker thereof. SPARCL1 is a matricellular protein expressed and secreted exclusively by the vascular system (endothelial and mural cells). In previous studies, gene expression analyses in CRC tumor tissues of different patient cohorts congruently showed a significant loss of SPARCL1 in colon and rectum adenocarcinomas as compared to uninvolved colon tissues and indicated that this loss is TME-dependent. Specifically, high SPARCL1 expression was associated with a Th1-TME, reduced incidence of distant metastasis and increased cancer-related survival. In contrast, SPARCL1 expression was lost in aggressive CRC with a non-Th1-TME. Here we show in functional analyses that SPARCL1 inhibits angiogenesis in different in vitro (endothelial cell proliferation, capillary formation on matrigel, 3D spheroid sprouting), ex vivo (fetal mouse metatarsal explant) and in vivo (FITC-dextran vascular permeability) assays. Moreover, increased tumor cell colonization is detected in the lungs of SPARCL1-knockout mice, further consolidating the anti-tumorigenic function of SPARCL1. To characterize the molecular mechanism of SPARCL1 anti-angiogenic function, we identified the TGF-β co-receptor Endoglin as a cellular receptor for SPARCL1 and found that SPARCL1 regulates ERK1/2 activity. The RAS-ERK pathway is amplified in more than half of all CRC tumors and as such is considered an important therapeutic target. Interestingly, SPARCL1 regulates ERK1/2 not only through phosphorylation but also through its subcellular localization. We show that SPARCL1 induces ERK1/2 phosphorylation preferentially in the cytoplasm and activates specifically cytoplasmic substrates downstream of it. Finally, we show that the regulation of ERK1/2 by SPARCL1 is not limited to endothelial cells but extends to epithelial CRC cells, indicative of autocrine and paracrine functions of SPARCL1. In summary, our study indicates that SPARCL1 is a TME-dependent angiocrine tumor suppressor in CRC acting through the regulation of ERK1/2 activity. This finding opens novel perspectives elucidating the crosstalk between the TME and the ERK signaling pathway and accordingly may enable new strategies to overcoming drug resistance and/or therapy-oriented patient stratification. Citation Format: Clara Tenkerian, Daniela Regensburger, Victoria Langer, Anika Klingler, Anne Borau, Heinrich Sticht, Andreas Ramming, Thomas Wohlfahrt, Benjamin Schmid, Valérie Méniel, Robert Grützmann, Nathalie Britzen-Laurent, Vera Schellerer, Michael Stürzl, Elisabeth Naschberger. SPARCL1 is an angiocrine inhibitor of tumorigenesis in colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 195.

Methylation of SPARCL1 Is Associated with Oncologic Outcome of Advanced Upper Urinary Tract Urothelial Carcinoma.

Advanced upper urinary tract urothelial carcinoma (UTUC) is often associated with poor oncologic outcomes. The secreted protein acidic and rich in cysteine-like 1 (SPARCL1) protein, belongs to the SPARC-related family of matricellular proteins. Much literature has been published describing the role of SPARCL1 in the prognosis many cancers. In this study, methylated promoter regions in high-grade and high-stage upper urinary urothelial tumours compared with normal urothelium were analyzed and revealed that SPARCL1 was the most significantly hypermethylated gene in UTUC tissues. Then we prospectively collected UTUC samples and adjacent normal urothelium for pyrosequencing validation, identifying significant CpG site methylation in UTUC tissues. In addition, SPARCL1 RNA levels were significantly lower in UTUC samples. Multivariate Cox regression analysis from 78 patients with solitary renal pelvic or ureteral pT3N0M0 urothelial carcinomas revealed that only negative SPARCL1 expression and nonpapillary tumour architecture were independently associated with systemic recurrence (p = 0.011 and 0.008, respectively). In vitro studies revealed that the behaviour of BFTC-909 cells was less aggressive and more sensitive to radiation or chemotherapy after SPARCL1 overexpression. Thus, SPARCL1 could be considered as a prognostic marker and help decision-making in clinical practice.

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Prognostic value of SPARCL1 in patients with colorectal cancer.

Secreted protein acidic and rich in cysteine-like 1 (SPARCL1), a member of extracelluar matrix glycoprotein, has been reported to be associated with various tumor types. The present study aimed to evaluate the prognostic value of SPARCL1 in patients with colorectal cancer. Tissue microarray blocks were constructed based on 79 patients who underwent radical surgery at the Kunshan First People's Hospital between 2008 and 2010. Thirty pairs of fresh-frozen tissues were also obtained for total protein extraction. The expression of SPARCL1 protein was analyzed using immunohistochemistry and western blotting analyses, and the association between overexpressed SPARCL1 and clinicopathological factors was evaluated. Survival analysis with Kaplan Meier curves and Cox regression analysis was used to analyze the prognostic value of SPARCL1. According to western blot analyses, SPARCL1 protein expression in colorectal tumors was significantly lower compared with corresponding normal tissues. The expression of SPARCL1 was markedly decreased from differentiation I to III, and the negative rate of SPARCL1 was higher at Duke's stage C compared with B. Though without any difference between ‘positive’ and ‘negative’ in overall survival, significantly higher survival in patients with positive SPARCL1 expression at Duke's stage B was detected in the present study. These results indicated that SPARCL1 may be a potential tumor suppressor gene and associated with good prognosis.

32P - SPARCL1, a novel prognostic predictive factor for GI malignancies: A meta-analysis

Background: Secreted protein acidic and rich in cysteines-like 1 (SPARCL1) is abnormally expressed in gastrointestinal (GI) malignancies. However, the correlation between SPARCL1 expression and the prognosis of patients remains unknown. Therefore, we performed a meta-analysis to investigate the potential value of SPARCL1 as a prognostic predictive marker for GI malignancies. Methods: The PubMed, Embase, EBSCO, CNKI, and Wanfang databases were systematically searched for studies examining SPARCL1 and clinicopathological features, including the prognoses of patients. Hazard ratios (HRs) and odds ratios (ORs) from individual studies were calculated and pooled using a random-effects or fix-effects model. Heterogeneity and publication bias analyses were performed. Results: Data from 8 studies, including a total of 2,356 patients, were summarized. The expression of SPARCL1 suggested a better prognosis (HR = 0.57, 95% CI: 0.445-0.698, P = 0.000) and was associated with clinicopathological features of GI malignancies, including distant metastasis (OR = 0.44, 95% CI: 0.23-0.85, P = 0.014), lymph node metastasis (OR = 0.56, 95% CI: 0.39–0.81, P = 0.002) and tumor differentiation (OR = 2.21, 95% CI: 1.82–2.69, P = 0.000). Subgroup analyses based on cancer type revealed that the expression of SPARCL1 had no effect on lymph node metastasis in colorectal cancer, and it did not influence tumor differentiation in gastric cancer. (all P > 0.05). Conclusions: SPARCL1 could be a novel prognostic predictive factor for GI malignancies. The expression of SPARCL1 could influence the clinicopathological features of GI malignancies. Further large-scale studies are essential to confirm SPARCL1’s prognostic predictive value, and more fundamental experimental studies are needed to illustrate the mechanisms. Legal entity responsible for the study: Hanguang HU Funding: None Disclosure: All authors have declared no conflicts of interest.

SPARCL1, a Novel Prognostic Predictive Factor for GI Malignancies: a Meta-Analysis

Background/Aims: Secreted protein acidic and rich in cysteines-like 1 (SPARCL1) is abnormally expressed in gastrointestinal (GI) malignancies. However, the correlation between SPARCL1 expression and the prognosis of patients remains unknown. Therefore, we performed a meta-analysis to investigate the potential value of SPARCL1 as a prognostic predictive marker for GI malignancies. Methods: The PubMed, Embase, EBSCO, CNKI, and Wanfang databases were systematically searched for studies examining SPARCL1 and clinicopathological features, including the prognoses of patients. Hazard ratios (HRs) and odds ratios (ORs) from individual studies were calculated and pooled using a random-effects or fix-effects model. Heterogeneity and publication bias analyses were performed. Results: Data from 8 studies, including a total of 2,356 patients, were summarized. The expression of SPARCL1 suggested a better prognosis (HR=0.57, 95% CI: 0.445-0.698, P=0.000) and was associated with clinicopathological features of GI malignancies, including distant metastasis (OR=0.44, 95% CI: 0.23-0.85, P=0.014), lymph node metastasis (OR=0.56, 95% CI: 0.39–0.81, P=0.002) and tumor differentiation (OR=2.21, 95% CI: 1.82–2.69, P=0.000). Subgroup analyses based on cancer type revealed that the expression of SPARCL1 had no effect on lymph node metastasis in colorectal cancer, and it did not influence tumor differentiation in gastric cancer. Egger’s test showed no evidence of publication bias (all P>0.05). Conclusion: SPARCL1 could be a novel prognostic predictive factor for GI malignancies. The expression of SPARCL1 could influence the clinicopathological features of GI malignancies. Further large-scale studies are essential to confirm SPARCL1’s prognostic predictive value, and more fundamental experimental studies are needed to illustrate the mechanisms.

Expression and role of SPARCL1 gene in tumor

Abnormal expression of secreted protein acidic and rich in cysteine like 1 (SPARCL1) gene is closely related to the development, metastasis and prognosis of a variety of tumors. Recent studies show that SPARCL1 gene is high expressed in glioma. The protein product of SPARCL1 gene encoded can interact with collagen, thus affecting the metastasis ability of tumor, which is positively correlated with tumor malignant degree. SPARCL1 gene is low expressed in gastric cancer, colorectal cancer, prostate cancer and breast cancer, which is negatively correlated with the invasion and metastasis abilities of tumors. At present, the role of speci-fic molecular mechanisms of SPARCL1 gene remains controversial. The expressions and functions of SPARCL1gene in tumor tissues seem to depend on the tumor microenvironment. Key words: Neoplasms; Neoplasm metastasis; Secreted protein acidic and rich in cysteine like 1 gene

SPARCL1-containing neurons in the human brainstem and sensory ganglion

Secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) is a member of the osteonectin family of proteins. In this study, immunohistochemistry for SPARCL1 was performed to obtain its distribution in the human brainstem, cervical spinal cord, and sensory ganglion. SPARCL1-immunoreactivity was detected in neuronal cell bodies including perikarya and proximal dendrites, and the neuropil. The motor nuclei of the IIIrd, Vth, VIth, VIIth, IXth, Xth, XIth, and XIIth cranial nerves and spinal nerves contained many SPARCL1-immunoreactive (-IR) neurons with medium-sized to large cell bodies. Small and medium-sized SPARCL1-IR neurons were distributed in sensory nuclei of the Vth, VIIth, VIIIth, IXth, and Xth cranial nerves. In the medulla oblongata, the dorsal column nuclei also had small to medium-sized SPARCL1-IR neurons. In addition, SPARCL1-IR neurons were detected in the nucleus of the trapezoid body and pontine nucleus within the pons and the arcuate nucleus in the medulla oblongata. In the cervical spinal cord, the ventral horn contained some SPARCL1-IR neurons with large cell bodies. These findings suggest that SPARCL1-containing neurons function to relay and regulate motor and sensory signals in the human brainstem. In the dorsal root (DRG) and trigeminal ganglia (TG), primary sensory neurons contained SPARCL1-immunoreactivity. The proportion of SPARCL1-IR neurons in the TG (mean ± SD, 39.9 ± 2.4%) was higher than in the DRG (30.6 ± 2.1%). SPARCL1-IR neurons were mostly medium-sized to large (mean ± SD, 1494.5 ± 708.3 μm2; range, 320.4–4353.4 μm2) in the DRG, whereas such neurons were of various cell body sizes in the TG (mean ± SD, 1291.2 ± 532.8 μm2; range, 209.3–4326.4 μm2). There appears to be a SPARCL1-containing sensory pathway in the ganglion and brainstem of the spinal and trigeminal nervous systems.

Androgen-Regulated SPARCL1 in the Tumor Microenvironment Inhibits Metastatic Progression.

Prostate cancer is a leading cause of cancer death in men due to the subset of cancers that progress to metastasis. Prostate cancers are thought to be hardwired to androgen receptor (AR) signaling, but AR-regulated changes in the prostate that facilitate metastasis remain poorly understood. We previously noted a marked reduction in secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) expression during invasive phases of androgen-induced prostate growth, suggesting that this may be a novel invasive program governed by AR. Herein, we show that SPARCL1 loss occurs concurrently with AR amplification or overexpression in patient-based data. Mechanistically, we demonstrate that SPARCL1 expression is directly suppressed by androgen-induced AR activation and binding at the SPARCL1 locus via an epigenetic mechanism, and these events can be pharmacologically attenuated with either AR antagonists or HDAC inhibitors. We establish using the Hi-Myc model of prostate cancer that in Hi-Myc/Sparcl1(-/-) mice, SPARCL1 functions to suppress cancer formation. Moreover, metastatic progression of Myc-CaP orthotopic allografts is restricted by SPARCL1 in the tumor microenvironment. Specifically, we show that SPARCL1 both tethers to collagen in the extracellular matrix (ECM) and binds to the cell's cytoskeleton. SPARCL1 directly inhibits the assembly of focal adhesions, thereby constraining the transmission of cell traction forces. Our findings establish a new insight into AR-regulated prostate epithelial movement and provide a novel framework whereby SPARCL1 in the ECM microenvironment restricts tumor progression by regulating the initiation of the network of physical forces that may be required for metastatic invasion of prostate cancer.

Abstract 2375: Endothelial cells isolated from colorectal carcinoma exhibit tumor microenvironment-dependent plasticity allowing the identification of SPARCL1 as a novel endothelial cell quiescence factor

Abstract It is well known that tumor cell heterogeneity and the tumor microenvironment (TME) contribute to tumorigenesis. In contrast, stromal cell heterogeneity and plasticity in response to different TMEs and its impact on tumorigenesis has not been conclusively investigated in human tumors, as yet. Here we analyzed TME-induced heterogeneity of tumor endothelial cells (TECs) in colorectal carcinoma (CRC). To this goal vital and highly pure TEC cultures were isolated from CRC with a Th1-like TME (Th1-TME) and from CRC with a non-Th1-like TME (Ctrl-TME). Tumors with a Th1-TME exhibited a significantly reduced angiogenic activity and were associated with an improved prognosis of CRC patients. Comparative transcriptome analyses of the established TEC cultures identified a cluster of genes which was homogeneously expressed in TECs from Th1-TME but significantly different in TECs from Ctrl-TME. The gene encoding secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) was identified to be most strongly up-regulated in TECs derived from a Th1-TME. We could confirm with in situ hybridization and immunohistochemistry that in normal colon and CRC tissues SPARCL1 expression is associated with endothelial cells (EC). Of note, SPARCL1 was consistently and highly expressed in normal colon, to a lower extent in CRC with a Th1-TME and low or absent in CRC tissues with a Ctrl.-TME. In cell culture SPARCL1 expression was absent in actively proliferating EC, but highly induced in EC reaching a confluent quiescent state. The SPARCL1 induction in confluent cells was reversible after the reduction of cell density. In functional studies SPARCL1 inhibited proliferation, migration and 3D sprouting of cultivated EC indicating potent anti-angiogenic activity. At the morphological level vessels in normal colon and in CRC with a Th1-TME were larger as compared to vessels in tumors with Ctrl-TME. Accordingly, SPARCL1 characterizes quiescent mature vessels that are larger as compared to rapidly sprouting microvessels. Altogether our results show that TECs isolated from CRC with different TMEs exhibit specific and differential long-term gene expression profiles. Characterization of the respective genes identified SPARCL1 as an anti-angiogenic gene associated with EC quiescence. These results demonstrate for the first time that human tumor endothelial cells retain differential TMEs by cellular memory allowing the identification of genes functionally imprinting the TME in these cellular sensors. Citation Format: Andrea Liebl, Elisabeth Naschberger, Vera S. Schellerer, Ute Schaal, Patrick Kölbel, Sandra Grenz, Nathalie Britzen-Laurent, Tilman T. Rau, Susanne Merkel, Barbara Dietel, Ludger Klein-Hitpass, Werner Hohenberger, Roland S. Croner, Michael Stürzl. Endothelial cells isolated from colorectal carcinoma exhibit tumor microenvironment-dependent plasticity allowing the identification of SPARCL1 as a novel endothelial cell quiescence factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2375. doi:10.1158/1538-7445.AM2015-2375