Secreted Protein Acidic And Cysteine Rich Research Articles

Abstract PO4-28-09: Upregulation of the immune checkpoint protein B7-H3 is associated with an immune suppressive microenvironment in progression from in situ to invasive lobular breast cancer

Abstract Invasive lobular breast cancer (ILC) is an understudied subtype of breast cancer characterized by late recurrence, metastasis to serosal surfaces including the peritoneum, and poor long-term outcomes. Lobular carcinoma in situ (LCIS) is a non-obligate precursor to ILC but is associated with a 30% increased risk of developing ILC. Therefore, understanding the underlying changes that occur in the invasive lobular cells as well as their tumor microenvironment (TME) during the transition from LCIS to ILC is critical for the development of novel therapeutic targets that could be used in early disease. To address this, we used combined spatial whole genome transcriptomics and a 97-protein proteomics assay to examine the spatial molecular profiles of coexistent LCIS and ILC. PanCK-positive tumor cells and panCK-negative stromal cells were segmented and analyzed separately for both RNA and protein. Despite the close spatial proximity of LCIS and ILC, there were notable differences in gene and protein expression in the tumor cells as well as the tumor microenvironment (TME). RNA profiling revealed significant upregulation of genes encoding essential components of the extracellular matrix, including type I, 3 and 5 collagens, small leucine-rich proteoglycans byglycan (BGN) and lumican (LUM), periostin (POSTN) and secreted protein acidic and cysteine rich (SPARC). Metalloprotease genes MMP2 and MMP11 were also upregulated. Genes highly expressed in the LCIS tumor compartment were KRT5, KRT14 KRT17 and MYLK which are highly expressed in myoepithelial cells at the periphery of the LCIS. One of the most upregulated proteins in ILC cells was B7 homology 3 protein (B7-H3) or Cluster of Differentiation 276 (CD276). B7-H3 is a transmembrane immunoregulatory protein belonging to the B7 family (that also includes PD-L1 and PD-L2). This immune checkpoint protein is overexpressed in many tumors but is barely expressed in normal cells. B7-H3 has been associated with several aspects of cancer progression, such as evasion of tumor immune surveillance and metastasis, and is strongly linked to poor prognosis in cancer. Compared to the LCIS TME, B7-H3 was also the most upregulated protein in the ILC TME along with the tumor-promoting M1 macrophage marker CD68 and the regulatory T-cell marker FOXP3. The lymphocyte marker CD45 was downregulated as well as T-cell proteins CD3, CD40 and granzyme B. Moreover, CD27, a memory B-cell marker was most highly expressed in the TME surrounding LCIS compared to ILC. Overall, these molecular profiles support a transition to a toward a much more immunosuppressive environment for invasive tumor cells compared to LCIS. Based on these data, our working hypothesis is that upregulation of B7-H3 on lobular tumor cells promotes invasion allowing interaction with laminins, integrins and other ECM proteins in the basement membrane and the interstitial membrane as LCIS acquire invasive properties of ILC. Furthermore, upregulation of B7-H3 on CD3+ HLA-DR+ expressing antigen presenting cells also promotes a suppressive immune microenvironment by inhibiting T-cell proliferation and downregulating cytokine production. In sum, these strongly concurrent spatial transcriptome and protein data suggest that B7-H3 and the immune suppressive network of ILC may be a promising target for early stage lobular breast cancer. B7-H3 Inhibitors could be explored in the neoadjuvant setting to evaluate the efficacy of immunotherapy in primary ILC. Citation Format: Lynda Bennett, Sunati Sahoo, Cheryl Lewis, Indu Raman, Candace Frerich, Guanchun Chen, Min Xu, Suzanne Conzen. Upregulation of the immune checkpoint protein B7-H3 is associated with an immune suppressive microenvironment in progression from in situ to invasive lobular breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-09.

Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity

BackgroundCancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.ResultsIn sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms.ConclusionThis work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

Therapeutic effects of in vivo administration of an inhibitor of tryptophan 2,3-dioxygenase (680c91) for the treatment of fibroids: a preclinical study

ObjectiveFibroids are characterized by marked overexpression of Tryptophan 2,3 dioxygenase (TDO2). The objective of this study was to determine the effectiveness of in vivo administration of an inhibitor of TDO2 (680C91) on fibroid size and gene expression. DesignAnimal and ex vivo human study. SubjectsSCID mice bearing human fibroid xenografts treated with vehicle and TDO2 inhibitor. InterventionDaily intraperitoneal administration of 680C91 or vehicle for two months and in vitro studies with fibroid explants. Main Outcome MeasuresTumor weight and gene expression profile of xenografts and in vitro mechanistic experiments using fibroid explants. ResultsCompound 680C91 was well-tolerated with no effects on blood chemistry and body weight. Treatment of mice with 680C91 resulted in 30% reduction in the weight of fibroid xenografts after two months of treatment and as expected lower levels of kynurenine, the byproduct of tryptophan degradation and an endogenous ligand of AhR (aryl hydrocarbon receptor) in the xenografts. The expression of CYP1B1 (cytochrome P450 family 1 subfamily B member 1), TGF-β3 (transforming growth factor beta 3), FN1 (fibronectin), CDK2 (cyclin-dependent kinase 2), E2F1 (E2F transcription factor 1), IL-8 (interleukin 8) and SPARC (secreted protein acidic and cysteine rich) mRNA, were lower in the xenografts of mice treated with 680C91 as compared with vehicle controls. Similarly, the protein abundance of collagen, FN1, CYP1B1 and SPARC were lower in the xenografts of 680C91 treated mice as compared with vehicle controls. Immunohistochemical analysis of xenografts indicated decreased expression of collagen, Ki67 and E2F1 but no significant changes in cleaved caspase 3 expression in mice treated with 680C91. The levels of kynurenine in the xenografts showed a direct correlation with the tumor weight and FN1 levels. In vitro studies with fibroid explants showed a significant induction of CYP1B1, TGF-β3, FN1, CDK2, E2F1, IL8 and SPARC mRNA by tryptophan which could be blocked by co-treatment with 680C91 and the AhR antagonist CH-223191. ConclusionThe results indicate that correction of aberrant tryptophan catabolism in fibroids could be an effective treatment through its effect to reduce cell proliferation and ECM accumulation.

SPARC Is a Novel Positive Immunohistochemical Marker of Epithelioid Mesothelioma to Differentiate It From Lung Adenocarcinoma and/or Squamous Cell Carcinoma.

Epithelioid mesothelioma with a solid histologic pattern (solid epithelioid mesothelioma) is difficult to distinguish from a poorly differentiated squamous cell lung carcinoma and/or solid lung adenocarcinoma. Thus, immunohistochemical markers are essential for diagnosis; however, the sensitivity and specificity of pre-existing mesothelial markers are suboptimal, particularly for differentiation from squamous cell carcinoma. Using a cancer-dependency map, we analyzed gene expression data of pleural mesothelioma and non-small cell lung cancer cell lines (squamous cell carcinoma and adenocarcinoma) and identified secreted protein acidic and cysteine-rich (SPARC) as a promising candidate for the differential diagnosis of epithelioid mesothelioma from lung squamous cell carcinoma and/or lung adenocarcinoma. SPARC expression in mesothelioma and lung cancer cell lines was validated using reverse-transcription polymerase chain reaction, western blotting, and immunohistochemistry. Immunohistochemical staining was performed using anti-SPARC antibodies against solid epithelioid mesothelioma, solid lung adenocarcinoma, and poorly differentiated lung squamous cell carcinoma. SPARC positivity was seen in 42/45 (93.3%) of solid epithelioid mesothelioma, 2/40 (5%) solid lung adenocarcinoma, and 2/45 (4.5%) of lung squamous cell carcinomas. The sensitivity, specificity, and diagnostic accuracy for differentiating solid epithelioid mesothelioma from lung cancer (solid lung adenocarcinoma and poorly differentiated lung squamous cell carcinoma) were 93.3, 95.2, and 94.6%, respectively. In conclusion, SPARC is a novel mesothelial marker that can be used to differentiate epithelioid mesothelioma from squamous cell carcinoma and lung adenocarcinoma.

Ischemia-Reperfusion Injury in Porcine Aortic Valvular Endothelial and Interstitial Cells.

Ischemia-reperfusion injury (IRI) in the myocardium has been thoroughly researched, especially in acute coronary syndrome and heart transplantation. However, our understanding of IRI implications on cardiac valves is still developing. This knowledge gap becomes even more pronounced given the advent of partial heart transplantation, a procedure designed to implant isolated human heart valves in young patients. This study aims to investigate the effects of IRI on aortic valvular endothelial cells (VECs), valvular interstitial cells (VICs), and whole leaflet cultures (no separation of VECs and VICs). We employed two conditions: hypoxic cold storage reperfusion (HCSR) and normothermia (NT). Key markers, secreted protein acidic and cysteine rich (SPARC) (osteonectin), and inducible nitric oxide synthase (iNOS2) were evaluated. In the isolated cells under HCSR, VICs manifested a significant 15-fold elevation in SPARC expression compared to NT (p = 0.0016). Conversely, whole leaflet cultures exhibited a 1-fold increment in SPARC expression in NT over HCSR (p = 0.0011). iNOS2 expression in VECs presented a marginal rise in HCSR, whereas, in whole leaflet settings, there was a 1-fold ascent in NT compared to HCSR (p = 0.0003). Minor escalations in the adhesion molecules intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), E-selection, and P-selection were detected in HCSR for whole leaflet cultures, albeit without statistical significance. Additionally, under HCSR, VICs released a markedly higher quantity of IL-6 and IL-8, with respective p-values of 0.0033 and <0.0001. Interestingly, the IL-6 levels in VECs remained consistent across both HCSR and NT conditions. These insights lay the groundwork for understanding graft IRI following partial heart transplantation and hint at the interdependent dynamic of VECs and VICs in valvular tissue.

Lung extracellular matrix modulates KRT5+ basal cell activity in pulmonary fibrosis

Aberrant expansion of KRT5+ basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5+ cells in IPF have not been delineated. Here, we reveal a potential mechanism by which KRT5+ cells migrate within the fibrotic lung, navigating regional differences in collagen topography. In vitro, KRT5+ cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry- based proteomics revealed compositional differences in ECM components secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrix restricts KRT5+ cell migration in vitro. Together, our findings demonstrate how changes to the ECM in IPF directly influence KRT5+ cell behaviour and function contributing to remodelling events in the fibrotic niche.

Impact of SPARC expression on treatment response of pembrolizumab and brain metastasis in patients with metastatic non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) often exhibits elevated Secreted Protein Acidic and Cysteine-Rich (SPARC) expression. In this study, we investigated the impact of SPARC expression on clinicopathologic features, pembrolizumab response, and prognosis in metastatic NSCLC patients. MethodsThirty-six patients diagnosed with metastatic NSCLC without actionable driver mutation and who received pembrolizumab with or without chemotherapy were included in this study. PD-L1 and SPARC expression were evaluated, with PD-L1 expression categorized based on tumor proportion score and SPARC staining intensity graded as 1+, 2+, and 3 +. Patients’ characteristics were compared across groups, and possible predictive markers were determined by binary logistic regression analysis. ResultsNo significant associations were found between SPARC expression and smoking status, histopathological tumor type, T and N status, and liver and bone metastasis. Higher SPARC expression was significantly linked to lower brain metastasis rates but higher CNS progression rates (p = 0.022 and p = 0.011, respectively. The objective response rate (ORR) showed a trend of being higher in the SPARC 1 + group (85.7% vs. 43.8% and 50.0% in 2 + and 3 + groups, respectively, p = 0.052. Univariate analysis did not find SPARC expression to be a significant prognostic factor for progression-free survival (PFS) (p = 0.7) and overall survival (OS) (p = 0.07).SPARC 1 + expression negatively affected the pembrolizumab response(p = 0.04,OR:0.11, 95%CI 0.01–0.92). ConclusionsOur study sheds light on a novel aspect of SPARC expression as a potential predictor of pembrolizumab response and a marker for CNS progression in metastatic NSCLC patients treated in the first-line setting.

Impact of SPARC expression on pembrolizumab treatment response and prognostic role in advanced stage non-small cell lung cancer.

e21042 Background: Secreted protein acidic and cysteine-rich (SPARC) was highly expressed in non-small cell lung cancer (NSCLC) tissue. Studies have indicated the association between SPARC expression and immune-cell infiltration and the prognosis of lung cancer. Despite the optimal first-line treatment approach in advanced NSCLC that lacks a driver mutation includes immunotherapy with or without chemotherapy, a well-defined tool or test for predicting response has yet to be created. We aimed to elucidate the influence of SPARC expression terms of clinicopathology, pembrolizumab response and prognosis in metastatic NSCLC patients. Methods: Thirty-six patients diagnosed with advanced-stage NSCLC without actionable driver mutation and recieved pembrolizumab in the first line setting were included in this study. Programmed death ligand-1(PD-L1) and SPARC expression were assessed by means of PD-L1 IHC 22C3 pharmDx and BenchMark ULTRA assay(Ventana Medical Systems, Tucson, AZ) in paraffin-embedded blocks. The expressions were categorised according to the tumor proportion score. Staining intensity of SPARC was graded according to the following criteria: 1+ (weak), 2+ (moderate), and 3+(strong). Results:The median age was 62 (range 45-81). One patient was female, and four patients did not have a smoking history.Twenty-seven of tumor were adenocarcinoma, and six were squamous cell carcinoma. PDL-1 status was &lt; 1% in 4 (11.4%), 1-49% in 12 (34.3%), and &gt; 50% in 20 (54.3%) patients.There was no significant correlation between SPARC expression and smoking status, histologic type of tumour, T and N status, and liver and bone metastasis. Central nervous system (CNS) metastasis and progression was seen in 8 (57.1%), 2 (12.5%), and 1 (16.7%) and in 6 (16.7%), 0, and 6 (37.5%) patients with SPARC 1+,2+, and 3 +, respectively. Controversially, higher SPARC expression was significantly correlated with lower rates of brain metastasis but higher rates of CNS progression (p = 0.022 and p = 0.011, respectively). While PD-L1 and SPARC expression were not significantly correlated, a trend toward low SPARC expression was seen in patients with PD-L1 &gt; 50%. The objective response rate (ORR) trended to be higher in the SPARC 1+ group (85.7%, 43.8%, and 50.0% in SPARC 1+, 2+ and 3+ group, respectively, p = 0.052). Univariate analysis revealed that SPARC expression was not a significant prognostic factor for both PFS (p = 0.7) and OS (p = 0.07). But, low SPARC expression was a significant predictive factor for the first-line pembrolizumab treatment response (p = 0.04, OR:0.11, 95%CI 0.01-0.92) in NSLC patients. Conclusions: Our study lightened a new area in the literature that SPARC expression might predict the pembrolizumab response and might be a marker for CNS progression in NSLC patients. These results should be explored in further studies to address the potential therapeutic implications of SPARC expression.

MiR-1275 Targeting SPARC Promotes Gambogic Acid-Induced Inhibition of Gastric Cancer.

Gambogic acid (GA) has been observed to effectively impede the progression of numerous types of cancers. In this study, we investigated the effects of miR-1275 and Secreted Protein Acidic and Cysteine Rich (SPARC) on GA in gastric cancer (GC). miR-1275 and SPARC expression were determined in GC cell lines and tissues using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The correlation between miR-1275 and SPARC expression was ascertained using Pearson's correlation coefficient. Cell proliferation was assessed using the cell counting kit-8 (CCK-8) assay. The Transwell assay was conducted to examine cell migration. A dual-luciferase reporter assay was used to verify the regulatory relationship between miR-1275 and SPARC. The levels of SPARC, Bcl-2, and Bax proteins were estimated using western blotting. To verify the effects of GA on the growth of GC cells in vivo, a tumorigenesis experiment was performed in nude mice. GA suppressed GC cell viability and migration, facilitated apoptosis, and inhibited tumor growth in vivo and in vitro. Low levels of miR-1275 been observed in GC cell lines and tissues. GA-treated GC cells manifested high miR-1275 levels. In functional experiments, miR-1275 enhanced the influence of GA on cell apoptosis, migration, and proliferation. Furthermore, GA treatment suppressed SPARC upregulation in GC cell lines and tissues. Pearson's correlation coefficient revealed that miR-1275 expression negatively correlated with SPARC expression. Mechanistically, miR-1275 promoted growth inhibition in GA-treated GC cells by targeting SPARC. Our study indicates that miR-1275 enhances the suppressive effect of GA on GC progression by inhibiting SPARC expression. Through this study, we contribute to the knowledge of a new mechanism by which GA suppresses GC progression.

Quantitative super-resolution imaging of platelet degranulation reveals differential release of von Willebrand factor and von Willebrand factor propeptide from alpha-granules

BackgroundVon Willebrand factor (VWF) and VWF propeptide (VWFpp) are stored in eccentric nanodomains within platelet alpha-granules. VWF and VWFpp can undergo differential secretion following Weibel-Palade body exocytosis in endothelial cells; however, it is unclear if the same process occurs during platelet alpha-granule exocytosis. Using a high-throughput 3-dimensional super-resolution imaging workflow for quantification of individual platelet alpha-granule cargo, we studied alpha-granule cargo release in response to different physiological stimuli. ObjectivesTo investigate how VWF and VWFpp are released from alpha-granules in response to physiological stimuli. MethodsPlatelets were activated with protease-activated receptor 1 (PAR-1) activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, secreted protein acidic and cysteine rich (SPARC), and fibrinogen were imaged using 3-dimensional structured illumination microscopy, followed by semiautomated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content. ResultsVWFpp overlapped with VWF in eccentric alpha-granule subdomains in resting platelets and showed a higher degree of overlap with VWF than SPARC or fibrinogen. Activation of PAR-1 (0.6-20 μM PAR-1 ap) or glycoprotein VI (GPVI) (0.25-1 μg/mL CRP-XL) signaling pathways caused a dose-dependent increase in alpha-granule exocytosis. More than 80% of alpha-granules remained positive for VWF, even at the highest agonist concentrations. In contrast, the residual fraction of alpha-granules containing VWFpp decreased in a dose-dependent manner to 23%, whereas SPARC and fibrinogen were detected in 60% to 70% of alpha-granules when stimulated with 20 μM PAR-1 ap. Similar results were obtained using CRP-XL. Using an extracellular anti-VWF nanobody, we identified VWF in postexocytotic alpha-granules. ConclusionWe provide evidence for differential secretion of VWF and VWFpp from individual alpha-granules.

Macromolecular crowding regulates matrix composition and gene expression in human gingival fibroblast cultures

Standard cell cultures are performed in aqueous media with a low macromolecule concentration compared to tissue microenvironment. In macromolecular crowding (MMC) experiments, synthetic polymeric crowders are added into cell culture media to better mimic macromolecule concentrations found in vivo. However, their effect on cultured cells is incompletely understood and appears context-dependent. Here we show using human gingival fibroblasts, a cell type associated with fast and scarless wound healing, that MMC (standard medium supplemented with Ficoll 70/400) potently modulates fibroblast phenotype and extracellular matrix (ECM) composition compared to standard culture media (nMMC) over time. MMC significantly reduced cell numbers, but increased accumulation of collagen I, cellular fibronectin, and tenascin C, while suppressing level of SPARC (Secreted Protein Acidic and Cysteine Rich). Out of the 75 wound healing and ECM related genes studied, MMC significantly modulated expression of 25 genes compared to nMMC condition. MMC also suppressed myofibroblast markers and promoted deposition of basement membrane molecules collagen IV, laminin 1, and expression of LAMB3 (Laminin Subunit Beta 3) gene. In cell-derived matrices produced by a novel decellularization protocol, the altered molecular composition of MMC matrices was replicated. Thus, MMC may improve cell culture models for research and provide novel approaches for regenerative therapy.

Formulating elafibranor and obeticholic acid with phospholipids decreases drug-induced association of SPARC to extracellular vesicles from LX-2 human hepatic stellate cells

Chronic hepatic diseases often compromise liver function and are directly responsible for up to two million yearly deaths world-wide. There are yet no treatment options to solve this global medical need.Experimental drugs elafibranor (Ela) and obeticholic acid (OA) appeared promising in numerous earlier studies, but they recently struggled to show significant benefits in patients. Little is known on the drugs’ impact on hepatic stellate cells (HSCs), key players in liver fibrogenesis. We recently reported a beneficial effect of polyenylphosphatidylcholines (PPCs)-rich formulations in reverting fibrogenic features of HSCs, including differences in their extracellular vesicles (EVs).Here, we newly formulated Ela and OA in PPC liposomes and evaluated their performance on the LX-2 (human HSC) cell line through our rigorous methods of EV-analysis, now expanded to include lipidomics. We show that direct treatments with Ela and OA increase EV-associated secreted protein acidic and cysteine rich (SPARC), a matricellular protein overexpressed in fibrogenesis. However, our results suggest that this potentially damaging drugs’ action to HSCs could be mitigated when delivering them with lipid-based formulations, most notably with a PPC-rich phospholipid inducing specific changes in the cellular and EV phospholipid composition. Thus, EV analysis substantially deepens evaluations of drug performances and delivery strategies.

Prognostic significance of SPARC expression in non-small cell lung cancer: A meta-analysis and bioinformatics analysis.

The aim of the present study was to elucidate the significance of secreted protein acidic and cysteine rich (SPARC) expression in non-small cell lung cancer (NSCLC) in terms of clinicopathology, immune-cell infiltration and survival prognosis. A meta-analysis and bioinformatics analysis were performed using studies retrieved with PubMed, Web of Science, Wanfang Data and the Chinese National Knowledge Infrastructure databases. The meta-analysis suggested that, compared with normal tissues, SPARC expression was elevated in NSCLC tissues. The expression of SPARC was not significantly associated with TNM stage and lymph-node metastasis, and was positively associated with patient gender. Regarding the differential expression of SPARC and the relationship between expression levels and survival, the Oncomine database was consulted and Kaplan-Meier curves were drawn. It was indicated that SPARC mRNA expression levels were higher in NSCLC tissues than in normal tissues. Low expression of SPARC mRNA was negatively associated with overall survival, first progression survival and post-progression survival of patients. Further exploration of the relationship between SPARC expression and survival by univariate analysis indicated that TNM stage, lymph node metastasis, distant metastasis and depth of infiltration of lung cancer were negatively associated with patient prognosis. Cox multifactorial analysis suggested that SPARC expression levels and TNM stage were risk factors significantly affecting the prognosis of patients with NSCLC. Analysis with the GEPIA and UALCAN databases further indicated that the mRNA expression level of SPARC in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) was higher than that in normal lung tissue, and the SPARC expression levels were affected by factors such as the TNM stage of lung cancer. A lower the level of SPARC mRNA expression was associated with a better relative survival prognosis of patients. In the Human Protein Atlas database, the expression level of SPARC protein was higher in LUAD and LUSC than in normal lung tissue. In the Timer database, the expression level of SPARC was closely linked to immune cells related to the occurrence of lung cancer, and the degree of immune-cell infiltration and SPARC protein expression were closely related to the prognosis of patients with lung cancer. Immune cells were indicated to exhibit significant inhibition of DNA proliferation mutation mechanisms in lung cancer (P<0.05). In summary, SPARC expression may be used as a valuable indicator of prognosis in patients with NSCLC, which may provide new approaches for preventative treatment.

A pH/H2 O2 /MMP9 Time-Response Gel System with Sparchigh Tregs Derived Extracellular Vesicles Promote Recovery After Acute Myocardial Infarction.

Regulatory T cells overexpressing SPARC (secreted protein acidic and cysteine rich) (Sparchigh Tregs) can help repair infarct tissues after acute myocardial infarction (AMI). This research demonstrates that Sparchigh Treg-derived extracellular vesicles (EVs) effectively improved cardiac function through proinflammatory factors IL-1β, IL-6, and TNF-α inhibition and collagen synthesis related gene Col3a1 promotion in AMI; moreover, a composite hydrogel-EVs system (DHPM(4APPC)_EVs) is designed based on Sparchigh Treg-derived EVs with CXCR2 overexpressing and pH/H2 O2 /MMP9 temporally responsive gel microspheres. In AMI, due to the levels of chemokine, pH, H2 O2 , and MMP9 enzymes in the infarct area, DHPM(4APPC)_EVs can effectively target the infarct area, release the loaded EVs, form the gel to capture the released EVs, and slowly release the captured EVs, contribute to promote EVs to stay in the infarct area for a long time to play the repair function, so as to reduce myocardial injury and promote the improvement of cardiac function. The proposed system in this research provides a potential approach for the treatment of AMI in the future.

Bevacizumab and sunitinib mediate osteogenic and pro-inflammatory molecular changes in primary human alveolar osteoblasts in vitro

Antiangiogenic medications target the de novo blood vessel formation in tumorigenesis. However, these novel drugs have been linked to the onset of medication-related osteonecrosis of the jaw (MRONJ). The aim of this in vitro study was to examine the effects of the vascular endothelial growth factor A (VEGFA) antibody bevacizumab (BEV) and the receptor tyrosine kinase inhibitor (RTKI) sunitinib (SUN) on primary human osteoblasts derived from the alveolar bone. Primary human alveolar osteoblasts (HAOBs) were treated with BEV or SUN for 48 h. Cellular metabolic activity was examined by XTT assay. Differentially regulated genes were identified by screening of 22 selected osteogenic and angiogenic markers by quantitative real-time reverse transcriptase polymerase chain reaction (qRT2-PCR). Protein levels of alkaline phosphatase (ALP), collagen type 1, α1 (COL1A1) and secreted protein acidic and cysteine rich (SPARC) were examined by enzyme-linked immunoassay (ELISA). Treatment with BEV and SUN did not exhibit direct cytotoxic effects in HAOBs as confirmed by XTT assay. Of the 22 genes examined by qRT2-PCR, four genes were significantly regulated after BEV treatment and eight genes in the SUN group as compared to the control group. Gene expression levels of ALPL, COL1A1 and SPARC were significantly downregulated by both drugs. Further analysis by ELISA indicated the downregulation of protein levels of ALP, COL1A1 and SPARC in the BEV and SUN groups. The effects of BEV and SUN in HAOBs may be mediated by alterations to osteogenic and catabolic markers. Therapeutic or preventive strategies in MRONJ may address drug-induced depression of osteoblast differentiation.

Possible Association Between Polymorphisms in ESR1, COL1A2, BGLAP, SPARC, VDR, and MMP2 Genes and Dental Fluorosis in a Population from an Endemic Region of West Bengal.

Dental fluorosis (DF) is the most prevalent form of fluorosis in India affecting millions of people all over the country. As estrogen receptor 1 (ESR1), collagen type 1 alpha 2 (COL1A2), bone γ-carboxyglutamic acid protein (BGLAP), secreted protein acidic and cysteine-rich (SPARC), vitamin D receptor (VDR), and matrix metallopeptidase 2 (MMP2) genes play critical roles in bone metabolism, bone formation, mineral metabolism, and mineralization, variants in these genes could influence susceptibility to DF. The present study was aimed at evaluating the association between 15 single-nucleotide polymorphisms (SNPs) in the six candidate genes (namely, ESR1, COL1A2, BGLAP, SPARC, VDR, and MMP2) and DF among 132 individuals (case = 71 and control = 61) living in a fluoride endemic region of West Bengal, India. No statistically significant association with disease risk was found when the genotypes and allele frequencies of each of the 15 SNPs was analyzed individually using odd's ratio with 95% confidence interval. "CC" and "AG" haplotypes of the COL1A2 gene showed a borderline association with DF. The present study is the first in India to use multifactor dimensionality reduction (MDR) analysis for identifying gene-gene and gene-environment interactions in fluorosis. The biomarker of serum fluoride showed a significant association with the disease state among the 17 attributes (15 SNPs and 2 biomarkers of urine fluoride and serum fluoride) (P value = 0.011). The best model of MDR analysis with maximized testing accuracy involved two SNPs from the ESR1 gene (rs9340799 and rs2077647) and one SNP from BGLAP gene (rs1543294) (P value < 0.0001).

Identification of Key Candidate Genes and Chemical Perturbagens in Diabetic Kidney Disease Using Integrated Bioinformatics Analysis.

Globally, nearly 40 percent of all diabetic patients develop serious diabetic kidney disease (DKD). The identification of the potential early-stage biomarkers and elucidation of their underlying molecular mechanisms in DKD are required. In this study, we performed integrated bioinformatics analysis on the expression profiles GSE111154, GSE30528 and GSE30529 associated with early diabetic nephropathy (EDN), glomerular DKD (GDKD) and tubular DKD (TDKD), respectively. A total of 1,241, 318 and 280 differentially expressed genes (DEGs) were identified for GSE30258, GSE30529, and GSE111154 respectively. Subsequently, 280 upregulated and 27 downregulated DEGs shared between the three GSE datasets were identified. Further analysis of the gene expression levels conducted on the hub genes revealed SPARC (Secreted Protein Acidic And Cysteine Rich), POSTN (periostin), LUM (Lumican), KNG1 (Kininogen 1), FN1 (Fibronectin 1), VCAN (Versican) and PTPRO (Protein Tyrosine Phosphatase Receptor Type O) having potential roles in DKD progression. FN1, LUM and VCAN were identified as upregulated genes for GDKD whereas the downregulation of PTPRO was associated with all three diseases. Both POSTN and SPARC were identified as the overexpressed putative biomarkers whereas KNG1 was found as downregulated in TDKD. Additionally, we also identified two drugs, namely pidorubicine, a topoisomerase inhibitor (LINCS ID- BRD-K04548931) and Polo-like kinase inhibitor (LINCS ID- BRD-K41652870) having the validated role in reversing the differential gene expression patterns observed in the three GSE datasets used. Collectively, this study aids in the understanding of the molecular drivers, critical genes and pathways that underlie DKD initiation and progression.

SPARC-positive macrophages are the superior prognostic factor in the microenvironment of diffuse large B-cell lymphoma and independent of MYC rearrangement and double-/triple-hit status

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with respect to outcome. Features of the tumor microenvironment (TME) are associated with prognosis when assessed by gene expression profiling. However, it is uncertain whether assessment of the microenvironment can add prognostic information to the most relevant and clinically well-established molecular subgroups when analyzed by immunohistochemistry (IHC). We carried out a histopathologic analysis of biomarkers related to TME in a very large cohort (n= 455) of DLBCL treated in prospective trials and correlated with clinicopathologic and molecular data, including chromosomal rearrangements and gene expression profiles for cell-of-origin and TME. The content of PD1+, FoxP3+ and CD8+, as well as vessel density, was not associated with outcome. However, we found a low content of CD68+ macrophages to be associated with inferior progression-free survival (PFS) and overall survival (OS; P= 0.023 and 0.040, respectively) at both univariable and multivariable analyses, adjusted for the factors of the International Prognostic Index (IPI), MYC break and BCL2/MYC and BCL6/MYC double-hit status. The subgroup of PDL1+ macrophages was not associated with survival. Instead, secreted protein acidic and cysteine rich (SPARC)-positive macrophages were identified as the subtype of macrophages most associated with survival. SPARC-positive macrophages and stromal cells directly correlated with favorable PFS and OS (both, P[log rank] <0.001, P[trend] < 0.001). The association of SPARC with prognosis was independent of the factors of the IPI, MYC double-/triple-hit status, Bcl2/c-myc double expression, cell-of-origin subtype and a recently published gene expression signature [lymphoma-associated macrophage interaction signature (LAMIS)]. SPARC expression in the TME detected by a single IHC staining with fair-to-good interobserver reproducibility is a powerful prognostic parameter. Thus SPARC expression is a strong candidate for risk assessment in DLBCL in daily practice.

Identification and evaluation of fructose-bisphosphate aldolase B as a potential diagnostic biomarker in choledochal cysts patients: a quantitative proteomic analysis.

BackgroundCholedochal cyst (CC) is a congenital bile duct malformation, with a higher incidence in minors. Patients with CCs are at risk of pancreatitis and ascending cholangitis. The main forms of treatments aim to avoid any possible hepatic, pancreatic, or biliary complications. Since early diagnosis is of great importance for CC treatment and prognosis, this investigation was designed to screen and identify potential biomarkers from the serum samples of CC patients for CC early diagnosis.MethodsQuantitative label free proteomic analysis was used to identify differentially expressed proteins in serum samples from CC patients and normal healthy children. The expression levels of biomarker candidates were further confirmed using quantitative polymerase chain reaction (Q-PCR), Western blot analysis, and immunohistochemistry in the choledochal tissues.ResultsThe quantitative label free proteomic analysis identified 47 differentially expressed proteins in the serum samples from the CC patients and the normal children, including 14 up-regulated proteins and 33 down-regulated proteins. The expression profile of eight biomarker candidates in CC patients, namely, insulin-like growth factor binding protein 2 (IGFBP2), tropomyosin (TPM3), fructose-bisphosphate aldolase B (ALDOB), fumarylacetoacetate hydrolase (FAH), superoxide dismutase 3 (SOD3), secreted protein acidic and cysteine rich (SPARC), apolipoprotein E (APOE), and retinol binding protein 4 (RBP4), were selected for further examination in choledochal tissues, showing that ALDOB was significantly increased.ConclusionsThe results demonstrated that the ALDOB protein increased significantly in choledochal tissues and the serum samples of CC patients, which may serve as an effective predictor for early diagnosis of CC.

High tumor cell platelet-derived growth factor receptor beta expression is associated with shorter survival in malignant pleural epithelioid mesothelioma.

Malignant pleural mesothelioma (MPM) has a rich stromal component containing mesenchymal fibroblasts. However, the properties and interplay of MPM tumor cells and their surrounding stromal fibroblasts are poorly characterized. Our objective was to spatially profile known mesenchymal markers in both tumor cells and associated fibroblasts and correlate their expression with patient survival. The primary study cohort consisted of 74 MPM patients, including 16 patients who survived at least 60 months. We analyzed location‐specific tissue expression of seven fibroblast markers in clinical samples using multiplexed fluorescence immunohistochemistry (mfIHC) and digital image analysis. Effect on survival was assessed using Cox regression analyses. The outcome measurement was all‐cause mortality. Univariate analysis revealed that high expression of secreted protein acidic and cysteine rich (SPARC) and fibroblast activation protein in stromal cells was associated with shorter survival. Importantly, high expression of platelet‐derived growth factor receptor beta (PDGFRB) in tumor cells, but not in stromal cells, was associated with shorter survival (hazard ratio [HR] = 1.02, p < 0.001). A multivariable survival analysis adjusted for clinical parameters and stromal mfIHC markers revealed that tumor cell PDGFRB and stromal SPARC remained independently associated with survival (HR = 1.01, 95% confidence interval [CI] = 1.00–1.03 and HR = 1.05, 95% CI = 1.00–1.11, respectively). The prognostic effect of PDGFRB was validated with an artificial intelligence‐based analysis method and further externally validated in another cohort of 117 MPM patients. In external validation, high tumor cell PDGFRB expression associated with shorter survival, especially in the epithelioid subtype. Our findings suggest PDGFRB and SPARC as potential markers for risk stratification and as targets for therapy.