scholarly journals 83 Spatially resolved transcriptomic and proteomic investigation of breast cancer and its immune microenvironment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A91-A91
Author(s):  
Jennifer Chew ◽  
Cedric Uytingco ◽  
Rapolas Spalinskas ◽  
Yifeng Yin ◽  
Joe Shuga ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Tumor Microenvironment ◽  
Protein Expression ◽  
Protein Detection ◽  
Response To Therapy ◽  
Pathological Examination ◽  
Multi Drug Resistance ◽  
Spatially Resolved ◽  
Gene And Protein Expression

BackgroundThe tumor microenvironment (TME) is composed of highly heterogeneous extracellular structures and cell types such as endothelial cells, immune cells, and fibroblasts that dynamically influence and communicate with each other. The constant interaction between a tumor and its microenvironment plays a critical role in cancer development and progression and can significantly affect a tumor’s response to therapy and capacity for multi-drug resistance. High resolution analyses of gene and protein expression with spatial context can provide deeper insights into the interactions between tumor cells and surrounding cells within the TME, where a better understanding of the underlying biology can improve treatment efficacy and patient outcomes. Here, we demonstrated the ability to perform streamlined multi-omic tumor analyses by utilizing the 10X Genomics Visium Spatial Gene Expression Solution for FFPE with multiplex protein enablement. This technique simultaneously assesses gene and protein expression to elucidate the immunological profile and microenvironment of different breast cancer samples in conjunction with standard pathological methods.MethodsSerial (5 µm) sections of FFPE human breast cancer samples were placed on Visium Gene Expression (GEX) slides. The Visium GEX slides incorporate ~5,000 molecularly barcoded, spatially encoded capture spots onto which tissue sections are placed, stained, and imaged. Following incubation with a human whole transcriptome, probe-based RNA panel and an immuno-oncology oligo-tagged antibody panel, developed with Abcam conjugated antibodies, the tissues are permeabilized and the representative probes are captured. Paired GEX and protein libraries are generated for each section and then sequenced on an Illumina NovaSeq at a depth of ~50,000 reads per spot. Resulting reads from both libraries are aligned and overlaid with H&E-stained tissue images, enabling analysis of both mRNA and protein expression. Additional analyses and data visualizations were performed on the Loupe Browser v4.1 desktop software.ConclusionsSpatial transcriptomics technology complements pathological examination by combining histological assessment with the throughput and deep biological insight of highly-multiplexed protein detection and RNA-seq. Taken together, our work demonstrated that Visium Spatial technology provides a spatially-resolved, multi-analyte view of the tumor microenvironment, where a greater understanding of cellular behavior in and around tumors can help drive discovery of new biomarkers and therapeutic targets.

50 Spatially resolved molecular investigation of triple negative breast cancer and its immune microenvironment

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A53-A53
Author(s):  
Stephen Williams ◽  
Cedric Uytingco ◽  
Neil Weisenfeld ◽  
Nigel Delaney ◽  
Solongo Ziraldo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Tumor Microenvironment ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Pathological Examination ◽  
Serial Sections ◽  
Rna Seq ◽  
Spatially Resolved ◽  
Whole Transcriptome

BackgroundTriple negative breast cancer (TNBC) accounts for 10–20% of all diagnosed breast cancer cases in the US and is characterized by loss of HER2, estrogen receptors, and progesterone receptors. TNBC is an aggressive, complex disease with a poor prognosis due to resistance to traditional therapies. Understanding the underlying biology and tumor microenvironment is critical to the development of diagnostic biomarkers and to guide the search for effective therapies. Here, we demonstrated the ability of the 10x Genomics Visium Spatial Gene Expression Solution to elucidate the immunological profile and microenvironment of TNBC samples in conjunction with standard pathological techniques.MethodsSpatial transcriptomics technology complement pathological examination by combining the benefits of histological stains with the throughput and deep biological insight of RNA-seq. We investigated serial sections of TNBC by using the 10x Genomics Visium Spatial Gene Expression Solution to spatially resolve the samples’ cellular composition and expressed microenvironment. Visium incorporates ~5000 molecularly barcoded, spatially encoded capture probes in spots over which a tissue section is placed and imaged. The samples are permeabilized and native mRNA is captured. Imaging and RNA sequencing data are processed together, resulting in whole transcriptome gene expression mapped to the tissue image.ResultsWe captured spatial patterns of gene expression and mapped the information back to H&E-stained images with regional annotations. Serial sections were then subject to fluorescence immunohistochemical staining for immune infiltrate paired with spatial gene expression capture. Subsequently, we combined these data with 3’ single-nuclei RNA-seq from the same tumor, generating expression profiles that were used to automatically annotate cell-types across the sections. This allowed for an understanding of the tumor microenvironment that could not be captured by image-based techniques alone. We resolved subgroups of spatially and biologically distinct immune, stem, and cancer progenitor cells. Finally, we digitally annotated tumor and normal tissue regions using expressed genetic mutations alone. Annotated tumor regions expressed more deleterious mutations than normal regions and we were able to automatically cluster regions of tumor vs. normal cells without any prior histopathological information. We also found intratumor gradients of mutational burden in oncogenes as well as non-cancer associated loci.ConclusionsTaken together, we demonstrated that Visium can provide a powerful complement to traditional histopathology, enabling both targeted panels and whole-transcriptome discovery of gene expression. This spatially resolved molecular information provides an unprecedented view into the tumor microenvironment and a powerful new tool for discovery of new biomarkers and therapeutic targets.

scholarly journals Differential expression between African-ancestry and White patients diagnosed with Triple-Negative Breast Cancer: EGFR, Myc, Bcl2 and β-Catenin as ancestry-associated markers

2020 ◽  
Author(s):  
Ana T. Matias ◽  
Ana Jacinta-Fernandes ◽  
Ana-Teresa Maia ◽  
Sofia Braga ◽  
António Jacinto ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
African American ◽  
Protein Expression ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
African Ancestry ◽  
Gene Set ◽  
Gene And Protein Expression ◽  
Differential Gene

AbstractPurposeTriple-negative breast cancer (TNBC) has a higher incidence, a younger age of onset, and a more aggressive behavior in African-ancestry women. Biological disparities have been suggested as an important factor influencing the ancestry-associated TNBC discrepancy. In this study, we sought to identify ancestry-associated differential gene and protein expression between African-ancestry and White TNBC patients, controlling for patients’ menopause status and pathological staging at diagnosis.MethodsDifferential gene expression analyses (DGEA) were performed using RNA-sequencing data from The Cancer Genome Atlas (TCGA). Gene set enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA), with focus on network design, were performed to highlight candidate genes for further validation through immunohistochemistry of TNBC samples from patients followed in Portugal.ResultsWith 52 African-American and 90 White TNBC patients included, TCGA’s data corroborate that African-American patients have a higher TNBC incidence (28.42% vs 11.89%, p<0.0001). Particularly, premenopausal and stage II disease African-American patients also have significantly lower survival probability, comparing with White patients (log-rank p=0.019 and 0.0038, respectively). DGEA results suggest that expression profile differences are more associated with TNBC staging than with patient’s menopause status. Hippo pathway and cellular community gene sets are downregulated, while breast cancer gene set is upregulated in African-Americans, comparing with White TNBC patients. Furthermore, MAPK pathway gene set is upregulated when controlling for stage II disease. Due to their central role in highly scored networks resulted from IPA’s network design, EGFR, Myc and Bcl2 genes were selected for further validation through immunohistochemistry. We also included β-Catenin in the validation study as it is consensually reported to be required in TNBC tumorigenesis. Although patients used in the DGEA and in the immunohistochemistry experiments are geographically and culturally distinct, both groups of African-ancestry patients are mostly of western-African ancestry and, interesting, differential gene and protein expression matched.ConclusionsWe found ancestry-associated gene expression patterns between African-ancestry and White TNBCs, particularly when controlling for menopause status or staging. EGFR, Myc, Bcl2 and β-catenin gene and protein differential expression matching results in distinct populations suggest these markers as being important indicators of TNBC’s ancestry-associated development.

Immune profiling and clinical outcomes in patients treated with ramucirumab and pembrolizumab in phase I study JVDF.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3089-3089 ◽  
Author(s):  
Roy S. Herbst ◽  
Hendrik-Tobias Arkenau ◽  
Emiliano Calvo ◽  
Johanna C. Bendell ◽  
Nicolas Penel ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Protein Expression ◽  
Clinical Outcomes ◽  
Related Gene ◽  
Immune Related Gene ◽  
Gene And Protein Expression ◽  
L1 Protein ◽  
Immune Profiling

3089 Background: In Study JVDF (NCT02443324), we combined ramucirumab (VEGFR2 antagonist) and pembrolizumab (PD-1 antagonist) to simultaneously target the tumor microenvironment and immune checkpoints in patients with advanced non-small cell lung cancer (NSCLC), gastric or gastroesophageal junction adenocarcinoma (G/GEJ), urothelial carcinoma (UC) or biliary tract cancer (BTC). We reported that this combination was associated with increased antitumor activity in patients with PD-L1 positive tumors by immunohistochemistry (IHC) compared with PD-L1 negative tumors.1) Here we explore the association between baseline gene expression profiles and clinical outcomes. Methods: JVDF was a nonrandomized phase 1a/b trial that treated patients with intravenous ramucirumab at 8 mg/kg on days 1 and 8 (G/GEJ, BTC) or 10 mg/kg on day 1 (G/GEJ, NSCLC, UC) plus pembrolizumab (200 mg day 1) every 3 weeks.1 Baseline tumor samples from 53 patients across 7 study cohorts were analyzed with the NanoString PanCancer Immune Profiling Panel for RNA expression and the DAKO PD-L1 IHC 22C3 pharmDx assay for PD-L1 protein expression. Clinical outcomes included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). Results: Across cohorts, PD-L1 gene expression was correlated with increased IFNγ gene expression and immune-related gene signatures (T effector, T cell-inflamed (TIS)), and trended with PD-L1 protein expression. Expression of immune checkpoint-related genes and myeloid-derived suppressor cell /regulatory T cell markers was increased in the NSCLC TPS≥50% PD-L1 IHC subgroup (N=8), while no clear pattern of expression was observed in other cohorts. Higher T effector and TIS scores appeared associated with better survival and response in NSCLC cohorts (mean TIS: 1.21±0.80 in responders (N=7) vs -0.13±0.57 in non-responders (N=7); p=0.004), and a trend was observed in G/GEJ cohorts (mean TIS: -0.18±0.30 (N=5) vs -0.39±0.21 (N=13); p=0.207). Of note, partial responses and stable disease were also observed in NSCLC and G/GEJ patients with a low baseline inflammatory signature score. Additional analyses are ongoing and will be presented. Conclusions: Baseline PD-L1 gene and protein expression tends to correlate with immune-related gene expression, and an inflamed tumor microenvironment may be associated with better clinical outcomes with ramucirumab and pembrolizumab. However, interpretation is limited by lack of control arm and sample size.1) Herbst et al. Lancet Oncol. 2019 Aug; 20 (8):1109-1123. Clinical trial information: NCT02443324 .

A gp78/AMFR protein-driven gene signature that predicts breast cancer outcome.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 553-553
Author(s):  
Sandeep K. Singhal ◽  
Jung Byun ◽  
Samson Park ◽  
Tingfen Yan ◽  
Ryan Yancey ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Protein Expression ◽  
Gene Expression Data ◽  
Independent Predictor ◽  
Response To Therapy ◽  
Expression Data ◽  
Rna Seq ◽  
Gene Expression Signatures ◽  
Gene Modules

553 Background: gp78, also known as the autocrine motility factor receptor (AMFR) or RNF45, is a polytopic RING-type E3 ubiquitin ligase resident to the endoplasmic reticulum (ER) that plays major role in the cellular response to stress by regulating ER homeostasis and signaling through its participation in the unfolded protein response (UPR) and ER associated degradation. We used machine learning (ML) and statistical modeling (SM) to assess gp78 as a protein biomarker that is an independent predictor of breast cancer (bc) survival exclusively in women of self-reported African descent as opposed to European ancestry. Methods: We examined a cohort of racially diverse 555 BC bc patients who underwent surgery for their primary BC in Greenville, NC using ML and SM approach. We leveraged the availability of RNA-seq gene expression data on a portion of our bc cohort (N=136 of 555) to construct gene expression signatures. Results: Using antibodies developed in the Weissman lab and established methods for quantitative IHC, we have found that gp78 expression is significantly increased in the tumors of bc patients compared to normal breast epithelia. In addition, we found that gp78 is expressed at significantly higher levels in bc of non-Hispanic black women (NHB) compared to non-Hispanic white women (NHW) (p=0.0038), and that bc subtypes known to be more aggressive and associated with higher grades like, Basal (p=1.6e-12), Luminal B (p=2.3e-4) and HER2(8.3e-4), display significantly higher levels of gp78 compare to Luminal A. Moreover, Kaplan-Meier survival curve analyses show that gp78 protein expression is more significantly associated with poor survival in NHB women (HR:1.65, p=0.073) compared to NHW women (HR:2.01, p=0.004). Finally, multivariate analysis reveals that gp78 protein expression, based on quantitative IHC, is an independent predictor of poor bc survival exclusively in women of African (NHB) ancestry (HR:1.99, p=0.017). We leveraged the availability of RNA-seq gene expression data on a portion of our bc cohort to construct gene expression signatures or gene modules. An analysis of pooled publicly available data from 845 patients that underwent neoadjuvant chemotherapy for bc (primarily taxane and anthracycline based), reveals that gp78 gene modules are highly predictive of patient response to therapy. gp78-derived gene modules show both high fold difference and significance in predicting response to therapy (AUC:0.72) which is very similar to other multi-gene panels that are currently in clinical use including Prosigna, MammaPrint, and Oncotype Dx. Conclusions: Our results show that gp78/AMFR is an independent predictor of bc survival and response to therapy, based on race, thus implicating a role for this protein, and potentially the UPR, as underlying biological differences in tumor properties linked to genetic ancestry.

scholarly journals Protein citrullination as a source of cancer neoantigens

2021 ◽  
Vol 9 (6) ◽  
pp. e002549
Author(s):  
Hiroyuki Katayama ◽  
Makoto Kobayashi ◽  
Ehsan Irajizad ◽  
Alejandro Sevillarno ◽  
Nikul Patel ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Immune Response ◽  
Protein Expression ◽  
Cell Lines ◽  
Cancer Cell ◽  
Family Members ◽  
Immunohistochemical Analysis ◽  
Cancer Cell Lines ◽  
Mhc Ii

BackgroundCitrulline post-translational modification of proteins is mediated by protein arginine deiminase (PADI) family members and has been associated with autoimmune diseases. The role of PADI-citrullinome in immune response in cancer has not been evaluated. We hypothesized that PADI-mediated citrullinome is a source of neoantigens in cancer that induces immune response.MethodsProtein expression of PADI family members was evaluated in 196 cancer cell lines by means of indepth proteomic profiling. Gene expression was assessed using messenger RNA data sets from The Cancer Genome Atlas. Immunohistochemical analysis of PADI2 and peptidyl-citrulline was performed using breast cancer tissue sections. Citrullinated 12–34-mer peptides in the putative Major Histocompatibility Complex-II (MHC-II) binding range were profiled in breast cancer cell lines to investigate the relationship between protein citrullination and antigen presentation. We further evaluated immunoglobulin-bound citrullinome by mass spectrometry using 156 patients with breast cancer and 113 cancer-free controls.ResultsProteomic and gene expression analyses revealed PADI2 to be highly expressed in several cancer types including breast cancer. Immunohistochemical analysis of 422 breast tumor tissues revealed increased expression of PADI2 in ER− tumors (p<0.0001); PADI2 protein expression was positively correlated (p<0.0001) with peptidyl-citrulline staining. PADI2 expression exhibited strong positive correlations with a B cell immune signature and with MHC-II-bound citrullinated peptides. Increased circulating citrullinated antigen–antibody complexes occurred among newly diagnosed breast cancer cases relative to controls (p=0.0012).ConclusionsAn immune response associated with citrullinome is a rich source of neoantigens in breast cancer with a potential for diagnostic and therapeutic applications.

scholarly journals Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Matthew Mannarino ◽  
Hosni Cherif ◽  
Li Li ◽  
Kai Sheng ◽  
Oded Rabau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Back Pain ◽  
Protein Expression ◽  
Disc Degeneration ◽  
Cell Senescence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Matrix Synthesis ◽  
Gene And Protein Expression ◽  
Pain Patients ◽  
In Cells

Abstract Background There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16INK4a, SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

scholarly journals Using cfRNA as a tool to evaluate clinical treatment outcomes in patients with metastatic lung cancers and other tumors

2021 ◽  
Author(s):  
Luis E. Raez ◽  
Kathleen Danenberg ◽  
Daniel Sumarriva ◽  
Joshua Usher ◽  
Jacob Sands ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Response To Therapy ◽  
Liquid Biopsies ◽  
Blood Samples ◽  
Lung Cancers ◽  
Metastatic Lung ◽  
Clinical Responses ◽  
Pre Treatment ◽  
Actin Expression

Aim: We report an exploratory analysis of cfRNA as a biomarker to monitor clinical responses in non-small cell lung cancer (NSCLC), breast cancer, and colorectal cancer (CRC). An analysis of cfRNA as a method for measuring PD-L1 expression with comparison to clinical responses was also performed in the NSCLC cohort. Methods: Blood samples were collected from 127 patients with metastatic disease that were undergoing therapy, 52 with NSCLC, 50 with breast cancer, and 25 with CRC. cfRNA was purified from fractionated plasma, and following reverse transcription (RT), total cfRNA and gene expression of PD-L1were analyzed by real-time polymerase chain reaction (qPCR) using beta-actin expression as a surrogate for relative amounts of cfDNA and cfRNA. For the concordance study of liquid biopsies and tissue biopsies, the isolated RNA was analyzed by RNAseq for the expressions of 13 genes. We had to close the study early due to a lack of follow-up during the Covid-19 pandemic. Results: We collected a total of 373 blood samples. Mean cfRNA PCR signals after RT were about 50-fold higher than those of cfDNA. cfRNA was detected in all patients, while cfDNA was detected in 88% of them. A high concordance was found for the expression levels of 13 genes between blood and solid tumor tissue. Changes in cfRNA levels followed over the course of treatments were associated with response to therapy, increasing in progressive disease (PD) and falling when a partial response (PR) occurred. The expression of PD-L1 over time in patients treated with immunotherapy decreased with PR but increased with PD. Pre-treatment levels of PD-L1 were predictive of response in patients treated with immunotherapy. Conclusion: Changes in cfRNA correlate with clinical response to the therapy. Total cfRNA may be useful in predicting clinical outcomes. PD-L1 gene expression may provide a biomarker to predict response to PD-L1 inhibition.

scholarly journals Metabolic clusters of breast cancer in relation to gene- and protein expression subtypes

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
Tonje H. Haukaas ◽  
◽  
Leslie R. Euceda ◽  
Guro F. Giskeødegård ◽  
Santosh Lamichhane ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Expression ◽  
Gene And Protein Expression

Angiopoietin 1 and 2 gene and protein expression is differentially regulated in acute anti-Thy1.1 glomerulonephritis

2008 ◽  
Vol 294 (5) ◽  
pp. F1174-F1184 ◽  
Author(s):  
Valentina Câmpean ◽  
Britta Karpe ◽  
Christian Haas ◽  
Akram Atalla ◽  
Harm Peters ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Glomerular Injury ◽  
Double Immunofluorescence ◽  
Real Time Quantitative Pcr ◽  
Glomerular Capillary ◽  
Gene And Protein Expression ◽  
Nonradioactive In Situ Hybridization ◽  
Angiopoietin 1

Capillary neoformation is important in repair of glomerular injury of various origins. VEGF was shown to be crucial for glomerular capillary repair in glomerulonephritis (GN). We reasoned that other angiogenic factors are likewise involved in glomerular capillary remodeling and found angiopoietin 1 and -2 (ANG1 and ANG2) mRNA to be upregulated in cDNA microarrays of microdissected glomeruli of anti-Thy1.1 GN of the rat. We then studied glomerular in situ gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 in the course of anti-Thy1.1 GN, which was induced by injection of OX-7 antibody. Animals were perfusion fixed at days 6 and 12 after GN induction and compared with nonnephritic controls receiving PBS. Capillary damage and repair were quantitatively analyzed using stereological techniques. Gene and protein expression of ANG1 and ANG2 and their receptor Tie-2 was analyzed using real-time quantitative PCR from microdissected glomeruli, nonradioactive in situ hybridization, double immunofluorescence, and Western blot analysis. Glomerular capillarization assessed as length density was significantly lower at day 6 of anti-Thy1.1 GN than in controls; it was back to normal values at day 12. ANG1 and ANG2 gene expression was markedly upregulated at day 6 of the disease compared with controls. Protein expression of ANG1 and ANG2 was confined to podocytes and that of Tie-2 to endothelial cells. At day 12 of anti-Thy1.1 GN when capillary restoration was nearly completed, ANG1 and ANG2 gene expression returned to basal levels, whereas Tie-2 expression was still high. With the use of a combined molecular and in situ approach, the spatial and temporal gene and protein expression of the angiopoietins and their receptor was analyzed in anti-Thy1.1 GN. The results indicate that glomerular expression of ANG1 and ANG2 and Tie-2 is differentially regulated and may contribute to healing and endothelial cell stabilization in experimental GN.

Effect of Auraptene on angiogenesis in Xenograft model of breast cancer

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mohammad Reza Shiran ◽  
Elham Mahmoudian ◽  
Abolghasem Ajami ◽  
Seyed Mostafa Hosseini ◽  
Ayjamal Khojasteh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Animal Model ◽  
Protein Expression ◽  
Western Blot ◽  
Xenograft Model ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Abstract Objectives Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. Methods In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1 × 104 HUVEC treated with Auraptene, 20 ng/mL EGF, 20 ng/mL bFGF and 20 ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Results Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80 μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Conclusions Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

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