Abstract
Simple SummaryOver 12% of women in the United States will be diagnosed with breast cancer in their lifetime. The overall 5-year survival rate for breast cancer is 90%, but the 5-year survival rate for women diagnosed with metastatic breast cancer is 28.1%. This study aims to characterize the cancerous cells that have left the primary tumor site and entered the blood, known as circulating tumor cells (CTCs). These cells could adhere to a site distant from the tumor and initiate metastasis. CTCs in breast cancer patients’ blood samples were enumerated and imaged. Cells from the blood were collected, RNA extracted, and the gene expression patterns of CTCs and other cell populations in the blood were investigated at the population and single cell level. This is a crucial step in characterizing CTCs as seeds of metastasis in breast cancer and for developing methods of detection to intercept metastasis before it localizes to distant regions of the body.Fatal metastasis occurs when circulating tumor cells (CTCs) disperse through the blood to initiate a new tumor at specific sites distant from the primary tumor. CTCs have been classically defined as nucleated cells positive for epithelial cell adhesion molecule and select cytokeratins (EpCAM/CK/DAPI), while negative for the common lymphocyte marker CD45. The enumeration of CTCs allows an estimation of the overall metastatic burden in breast cancer patients, but challenges regarding CTC heterogeneity and metastatic propensities persist, and their decryption could improve therapies. CTCs from metastatic breast cancer (mBC) patients were captured using the RareCyteTM Cytefinder II platform. The Lin− and Lin+ (CD45+) cell populations isolated from the blood of three of these mBC patients were analyzed by single-cell transcriptomic methods, which identified a variety of immune cell populations and a cluster of cells with a distinct gene expression signature, which includes both cells expressing EpCAM/CK (“classic” CTCs) and cells possessing an array of genes not previously associated with CTCs. This study put forward notions that the identification of these genes and their interactions will promote novel areas of analysis by dissecting properties underlying CTC survival, proliferation, and interaction with circulatory immune cells. It improves upon capabilities to measure and interfere with CTCs for impactful therapeutic interventions.
Highlights
Breast cancer is the most common cancer in women, accounting for 15% of all new cancer cases in the US
The detection of circulating tumor cells (CTCs) as defined by the epithelial-cell adhesion molecule (EpCAM)+/CK+/DAPI+/CD45− expression pattern has been observed in the blood of metastatic cancer patients, including metastatic breast cancer (mBC), and their relevance affecting metastatic competence has been established [16,21,45,46,47,48]
Patient blood samples were collected into sodium-EDTA tubes, and each sample was sub-divided. (a) One portion of the blood sample was processed for isolating Lin−/Lin+ cell populations via FACs, followed by RNA Sequencing or scRNA-Sequencing
Summary
Breast cancer is the most common cancer in women, accounting for 15% of all new cancer cases in the US. Primary breast tumors are typed according to hormonal receptor status (estrogen receptor (ER), progesterone receptor (PR), and Erb-B2 receptor tyrosine kinase 2 (ERBB2 or HER2) amplification); and in some cases, the presence of specific mutations or changes in gene copy number help to determine treatment. As the primary tumor grows, heterogeneous cell subpopulations with distinct gene expression signatures and biological characteristics arise. Some of these characteristics may modulate cellular adhesion and migration away from the tumor, as either a single cell or a cluster of cells. Because CTCs express distinct and specific transcriptional profiles compared to primary or metastatic tumors, they offer the unique opportunity to analyze transcriptional information, which can be therapeutically useful [7,13,14,15]
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