Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

IF 3.7 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Biological Procedures Online Pub Date : 2022-06-04 DOI:10.1186/s12575-022-00167-x

Ivanović, Vesna, Dedović-Tanić, Nasta, Milovanović, Zorka, Stojiljković, Bratislav, Demajo, Miroslav, Mandušić, Vesna

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引用次数: 0

Abstract

Metastatic Axillary Lymph Node (mALN) status is currently the most important prognostic factor in the management of primary breast cancer (BC). Thus, development of specimens which enable identification of new mALN markers, involved in the progression of the disease, are of considerable interest. The specific aim of this work was to describe the method of establishment of Metastatic Axillary Nodal Cell Suspension and its fractionation, termed Fractionated Nodal Cell Suspension (FNCS), into nuclear and cytosolic extracts to enable determination of protein expression levels of nuclear cFOS and cytosolic Transforming Growth Factor β1 (TGFβ1) in BC patients. To standardize the procedure, HeLa cells were successfully fractionated into nuclear/cytosolic extracts with confirmed presence of nuclear cFOS and cytosolic TGFβ1 proteins. Subsequently, the ALN Cell Suspension specimens were obtained and further fractionated from a pilot sample of six ALN tissue pairs, mALN versus autologous normal ALN (nALN), dissected from invasive BC patients. The mALN/nALN results revealed overexpression of both nuclear cFOS and cytosolic TGFβ1 protein levels. However, only the TGFβ1 data exhibited statistically significant overexpression, which was proportional to the respective values of mALN diameter of tumor deposits. Detailed protocol for establishment and fractionation of mALN cell suspension specimens, termed FNCS, into nuclear and cytosolic extracts is here described for the first time. This approach might be a convenient ex vivo model for simultaneous analysis of protein, RNA and DNA biomarkers from nuclear/cytosolic extracts of the same mALN tissue sample. It might have potential to enable, in the age of genomics and personalized medicine, an identification of novel mALN biomarkers and thus improve the screening, diagnosis and prognosis of invasive BC.

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转移性腋窝淋巴结细胞悬液的建立及分离检测乳腺癌患者核cFOS和胞质tgf - β1蛋白表达水平

转移性腋窝淋巴结(mALN)状态是目前原发性乳腺癌(BC)治疗中最重要的预后因素。因此，开发能够识别与疾病进展有关的新的mALN标记物的标本具有相当大的意义。这项工作的具体目的是描述转移腋窝淋巴结细胞悬液的建立及其分离的方法，称为分离淋巴结细胞悬液(FNCS)，成核和细胞质提取物，以测定BC患者核cFOS和细胞质转化生长因子β1 (tgf - β1)的蛋白表达水平。为了使操作标准化，成功地将HeLa细胞分离成核/细胞质提取物，并确认存在核cFOS和细胞质tgf - β1蛋白。随后，从浸润性BC患者解剖的6对ALN组织(mALN和自体正常ALN)的试点样本中获得ALN细胞悬液标本，并进一步分离。mALN/nALN结果显示核cFOS和胞质tgf - β1蛋白水平均过表达。然而，只有tgf - β1数据显示有统计学意义的过表达，这与肿瘤沉积物的mALN直径各自的值成正比。详细的方案建立和分离的mALN细胞悬浮液标本，称为FNCS，成核和细胞质提取物在这里首次描述。该方法可能是一种方便的离体模型，用于同时分析来自同一mALN组织样本的核/细胞质提取物的蛋白质，RNA和DNA生物标志物。在基因组学和个性化医疗的时代，它可能有潜力鉴定新的mALN生物标志物，从而改善浸润性BC的筛查、诊断和预后。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biological Procedures Online 生物-生化研究方法

CiteScore

10.50

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： iological Procedures Online publishes articles that improve access to techniques and methods in the medical and biological sciences. We are also interested in short but important research discoveries, such as new animal disease models. Topics of interest include, but are not limited to: Reports of new research techniques and applications of existing techniques Technical analyses of research techniques and published reports Validity analyses of research methods and approaches to judging the validity of research reports Application of common research methods Reviews of existing techniques Novel/important product information Biological Procedures Online places emphasis on multidisciplinary approaches that integrate methodologies from medicine, biology, chemistry, imaging, engineering, bioinformatics, computer science, and systems analysis.