scholarly journals Single-Cells Isolation and Molecular Analysis: Focus on HER2-Low CTCs in Metastatic Breast Cancer

Cancers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 79
Author(s):  
Paolo D’Amico ◽  
Carolina Reduzzi ◽  
Wenan Qiang ◽  
Youbin Zhang ◽  
Lorenzo Gerratana ◽  
...  

Although the detection of CTCs expressing HER2 at low intensity (HER2-low CTCs) has been shown to have a negative prognostic value in metastatic breast cancer (MBC) patients, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges behind the selective collection of immunophenotype-specific CTCs, we developed a pipeline to individually capture HER2-low CTCs. Four different breast cancer cell lines (MDA-MB-231, T47D, MDA-MB-453, and SKBR3), that are known to express HER2 at different immunohistochemistry levels (respectively classified as 0, 1+, 2+, and 3+), were spiked in healthy donor blood tubes (7.5 mL) and processed with the CellSearch® (Menarini Silicon Biosystems, Bologna, Italy) for enrichment and the DEPArray NxT™ for single cell selection. The HER2 signal-intensities of each cell line was compared using the nonparametric Mann–Whitney U test. The optimal cut-offs to distinguish HER2 1+ from 0 and 2+ cells were calculated performing the Receiver operating characteristic (ROC) curve. Median HER2 signal-intensities detected with the DEPArray NxT™ were: 2.59 (0), 3.58 (1+), 5.23 (2+) and 38.37 (3+). DEPArray NxT efficiently differentiated each single cell line (p < 0.001). The area under the ROC curve was 0.69 and 0.70 (respectively 0 vs. 1+ and 1+ vs. 2+) and the optimal calculated cut-offs were 2.85 (lower) and 4.64 (upper). HER2-low CTCs can be detected and separately collected using predetermined intensity cut-offs. This study will allow standardized single-cell or pooled collection of HER2-low CTCs for downstream molecular analyses.

2019 ◽  
Vol 23 (11n12) ◽  
pp. 1440-1447 ◽  
Author(s):  
Azusa Terasaki ◽  
Hiromi Kurokawa ◽  
Hiroko P. Indo ◽  
Hiroko Bando ◽  
Hisato Hara ◽  
...  

Breast cancer is one of the most common types of cancers prevalent in women. Several types of breast cancers can easily metastasize to bone and cause disease complications such as hypercalcemia and pathologic fracture, thus compromising the quality of life of people affected by it. Bisphosphonate drugs are often used for the treatment of bone metastasis to suppress osteoclastic bone resorption. However, bisphosphonate has adverse effects on the gastrointestinal tract and kidneys and also induces osteonecrosis of the jaw. Photodynamic therapy (PDT) is an alternative cancer treatment approach with minimal invasiveness. It is a combination treatment that uses photosensitizers, which accumulate in tumor cells, followed by laser irradiation. We previously reported that the cellular incorporation of 5-aminolevulinic acid (5-ALA), which was a precursor of protoporphyrin IX (PpIX), was regulated by reactive oxygen species derived from mitochondria (mitROS). In this study, we investigated the incorporation of 5-ALA, accumulation of PpIX, and subsequent effects on cell viability after laser irradiation of two different breast cancer cell lines with different metastaticites. The highly metastatic breast cancer cell line 4T1E/M3 showed a significant increase in ROS production after treatment with indomethacin (IND). In addition, IND treatment enhanced the cellular uptake of 5-ALA via PEPT1 upregulation in 4T1E/M3, but not in the non-metastatic cell line. Overall, metastatic breast cancer is likely to be sensitive to ROS and activate signaling pathways associated with 5-ALA transportation, suggesting that ALA-PDT could be an effective treatment with low invasiveness for metastatic breast cancer.


2021 ◽  
Author(s):  
Karla Helvie ◽  
Laura DelloStritto ◽  
Lori Marini ◽  
Nelly Oliver ◽  
Miraj Patel ◽  
...  

This standard operating procedure was established by the Center for Cancer Genomics at Dana-Farber Cancer Institute, the Brigham and Women's Hospital and the Klarman Cell Observatory at the Broad Institute, to standardize the collection of fresh metastatic breast cancer biopsies and their allocation to various bulk and single cell assays, including whole exome and bulk RNA-sequencing, single-cell RNA sequencing, and spatial profiling of RNA and protein. The use of a well defined workflow has allowed us to generate high quality data from these different assays, by implementing efficient modes of communication, minimizing the time elapsed from sample collection to preservation or processing, and ensuring optimal transportation conditions. Visual Abstract


2018 ◽  
Vol 5 (12) ◽  
pp. 1801158 ◽  
Author(s):  
Jialang Zhuang ◽  
Yongjian Wu ◽  
Liang Chen ◽  
Siping Liang ◽  
Minhao Wu ◽  
...  

2014 ◽  
Vol 9 (4) ◽  
pp. 749-757 ◽  
Author(s):  
Marta Pestrin ◽  
Francesca Salvianti ◽  
Francesca Galardi ◽  
Francesca De Luca ◽  
Natalie Turner ◽  
...  

Nature ◽  
2015 ◽  
Vol 526 (7571) ◽  
pp. 131-135 ◽  
Author(s):  
Devon A. Lawson ◽  
Nirav R. Bhakta ◽  
Kai Kessenbrock ◽  
Karin D. Prummel ◽  
Ying Yu ◽  
...  

2018 ◽  
Vol 6 (4) ◽  
pp. 349-361
Author(s):  
Yu-Chun Lin ◽  
Dong-Qing Chin

Understanding the molecular pathways that contribute to the development of metastatic breast cancer invasion and metastasis to brain is needed to improve the clinical utility of novel agents, and to predict the success of targeted personalized therapy based on tumor-specific mutations. Little is known about the clinical significance of HER2/ERK1/2/MMP-9 signaling pathway in breast cancer. We performed Global exon array to study the expression of ERK1/2/MMP-9 signaling pathway in metastatic breast cancer to brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of ERK1/2/MMP-9 proteins in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells.


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